Compositions for gut health

ABSTRACT

Provided herein, inter alia, are compositions of organic acid-producing microorganisms and methods of making and using the same to inhibit pathogenic bacterial populations in the gastrointestinal tracts of an animal and additionally promote improvement of one or more metrics in an animal, such as increased bodyweight gain, decreased feed conversion ratio (FCR). improved gut barrier integrity, reduced mortality, reduced pathogen infection, and reduced pathogen shedding in feces.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/887,847, filed Aug. 16, 2019, the disclosure of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

Provided herein, inter alia, are multi-strain direct fed microbialbacterial consortia useful for improving animal gut health and/orperformance as well as methods of making and using the same.

BACKGROUND

In monogastric animal species such as birds, the gastrointestinal tractand intestinal-associated microflora are not only involved in digestionand absorption but also interact with the immune and central nervoussystem to modulate health. The inside of the intestinal tract is coatedwith a thin layer of sticky, viscous mucous, and embedded in this mucuslayer are millions and millions of bacteria and other microbes. When theintestinal bacteria are in balance (i.e., the good bacteria outnumberthe bad bacteria), the gut is said to be healthy. A healthy microbiotaprovides the host with multiple benefits, including colonizationresistance to a broad spectrum of pathogens, essential nutrientbiosynthesis and absorption, and immune stimulation that maintains ahealthy gut epithelium and an appropriately controlled systemicimmunity. In settings of “dysbiosis” or disrupted symbiosis, microbiotafunctions can be lost or deranged, resulting in increased susceptibilityto pathogens, altered metabolic profiles, or induction ofproinflammatory signals that can result in local or systemicinflammation or autoimmunity. Thus, the intestinal microbiota of poultryplays a significant role in the pathogenesis of many diseases anddisorders, including a variety of pathogenic infections of the gut suchas coccidiosis or necrotic enteritis.

Over the past several years, there has been increasing governmental andconsumer pressure applied to the animal feed industry to decrease orcurtail the use of antibiotics as components of animal nutrition feedingregimens. This pressure is due in large part to the recognition that useof such antibiotics contribute to the rise of antibiotic-resistantpathogenic microorganisms. However, this “No Antibiotics Ever” consumertrend, especially in the poultry industry, has led to the re-emergenceof bacterial diseases, particularly necrotic enteritis (Poultry Science,Volume 97, Issue 6, 1 Jun. 2018, 1929-1933). Necrotic enteritis iscaused by certain toxin-producing Clostridium perfringens strains. Undercertain conditions C. perfringens produces toxins which cause lesions inthe small intestines and ultimately result in reduced growth or death ofthe infected birds. Accordingly, there is currently a recognized needfor products and methods capable of reducing pathogenic bacterialpopulations in the digestive tracts of domesticated animals such asbirds without the use of traditionally-used antibiotics.

The subject matter disclosed herein addresses these needs and providesadditional benefits as well.

SUMMARY

Provided herein, inter alia, are multi-strain direct fed microbialbacterial consortia of organic acid-producing microorganisms and methodsof making and using the same to inhibit pathogenic bacterial populationsin the gastrointestinal tracts of an animal (such as birds, for example,chickens) and additionally promote improvement of one or more metrics inan animal such as increased bodyweight gain, decreased feed conversionratio (FCR), improved gut barrier integrity, reduced mortality, reducedpathogen infection, and reduced pathogen shedding in feces.

Accordingly, in some aspects, provided herein is a feed additivecomposition comprising a direct fed microbial (DFM) comprising (a) atleast one biologically pure strain of (i) Lactobacillus reuteri; and/or(ii) L. salivarius; and (b) one or more additional biologically purestrain(s) of (i) L. reuteri: (ii) L. agilis; (iii) L. crispatus; and/or(iv) L. gallinarum. In some embodiments, the feed additive compositioncomprises three biologically pure strains of L. reuteri. In someembodiments of any of the embodiments provided herein, the feed additivecomposition comprises (a) a bacterial strain having a 16S ribosomal RNAsequence displaying at least 97.0% sequence similarity to a 16Sribosomal RNA sequence of a L. reuteri strain S1 deposited at WesterdijkFungal Biodiversity Institute (WFDB) under number CBS 145921; and (b)(i)a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.reuteri strain S2 deposited at WFDB under number CBS 145922; and (ii) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.reuteri strain S3 deposited at WFDB under number CBS 145923. In someembodiments of any of the embodiments provided herein, the compositioncomprises (a) L. reuteri strain S1 (CBS 145921) or a live strain havingall of the identifying characteristics of L. reuteri strain S1 (CBS145921); and (b)(i) L. reuteri strain S2 (CBS 145922) or a live strainhaving all of the identifying characteristics of L. reuteri strain S2(CBS 145922); and (ii) L. reuteri strain S3 (CBS 145923) or a livestrain having all of the identifying characteristics of L. reuteristrain S3 (CBS 145923) either (A) alone; or (B) in combination with aculture supernatant derived from each of these strains. In someembodiments, the feed additive composition comprises (a) a biologicallypure strain of L. salivarius; and (b)(i) a biologically pure strain ofL. gallinarum; and (ii)(A) a biologically pure strain of L. agilis; or(B) a biologically pure strain of L. reuteri. In some embodiments of anyof the embodiments provided herein, the feed additive compositioncomprises (a) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of a L. salivarius strain H2 deposited at WFDB under number CBS145919; and (b)(i) a bacterial strain having a 16S ribosomal RNAsequence displaying at least 97.0% sequence similarity to a 16Sribosomal RNA sequence of a L. gallinarum strain H1 deposited at WFDBunder number CBS 145918; and (ii)(A) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of an L. agilis strain 113 comprising SEQID NO:1; or (B) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of an L. reuteri strain A2 deposited at WFDB under number CBS145924. In some embodiments, the composition comprises (a) L. salivariusstrain H2 (CBS 145919) or a live strain having all of the identifyingcharacteristics of L. salivarius strain H2 (CBS 145919); and (b)(i) L.gallinarum strain H1 (CBS 145918) or a live strain having all of theidentifying characteristics of L. gallinarum strain H1 (CBS 145918); and(ii) L. reuteri strain A2 (CBS 145924) or a live strain having all ofthe identifying characteristics of L. reuteri strain A2 (CBS 145924)either (A) alone; or (B) in combination with a culture supernatantderived from each of these strains. In some embodiments, the feedadditive composition comprises (a) a biologically pure strain of L.salivarius; and (b)(i) a biologically pure strain of L. agilis; and abiologically pure strain of L. reuteri. In some embodiments of any ofthe embodiments provided herein, the feed additive composition comprises(a) a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.salivarius strain A1 comprising SEQ ID NO:2; and (b)(i) a bacterialstrain having a 16S ribosomal RNA sequence displaying at least 97.0%sequence similarity to a 16S ribosomal RNA sequence of an L. agilisstrain A3 comprising SEQ ID NO:3; and (ii) a bacterial strain having a16S ribosomal RNA sequence displaying at least 97.0% sequence similarityto a 16S ribosomal RNA sequence of an L. reuteri strain A2 deposited atWFDB under number CBS 145924 either (A) alone; or (B) in combinationwith a culture supernatant derived from each of these strains. The feedadditive composition of claim 9, comprising L. reuteri strain A2 (CBS145924) or a live strain having all of the identifying characteristicsof L. reuteri strain A2 (CBS 145924). The feed additive composition ofclaim 1, comprising (a) a biologically pure strain of L salivarius; and(b)(i) a biologically pure strain of L. agilis: and (ii) a biologicallypure strain of L. crispatus. In some embodiments of any of theembodiments provided herein, the feed additive composition comprises (a)a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.salivarius strain D2 comprising SEQ ID NO:4; (b)(i) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of an L. agilis strain D1comprising SEQ ID NO:5; and (ii) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of an L. crispatus strain D3 comprising SEQID NO:6 either (A) alone; or (B) in combination with a culturesupernatant derived from each of these strains. In some embodiments ofany of the embodiments provided herein, a) the 16S ribosomal RNAsequence of L. reuteri strain S1 comprises the nucleotide sequence ofSEQ ID NO:7; and (b)(i) the 16S ribosomal RNA sequence of L. reuteristrain S2 comprises the nucleotide sequence of SEQ ID NO:8; and (ii) the16S ribosomal RNA sequence of L. reuteri strain S3 comprises thenucleotide sequence of SEQ ID NO:9. In some embodiments of any of theembodiments provided herein, a) the 16S ribosomal RNA sequence of L.salivarius strain H2 comprises the nucleotide sequence of SEQ ID NO:10;and (b)(i) the 16S ribosomal RNA sequence of L. gallinarum strain H1comprises the nucleotide sequence of SEQ ID NO:11; and (ii) the 16Sribosomal RNA sequence of L. reuteri strain A2 comprises the nucleotidesequence of SEQ ID NO:12. In some embodiments, (b)(ii) the 16S ribosomalRNA sequence of L. reuteri strain A2 comprises the nucleotide sequenceof SEQ ID NO:12. In some embodiments of any of the embodiments providedherein, said one or more strains further comprise one or moreinactivated or deleted antimicrobial resistance (AMR) genes. In someembodiments of any of the embodiments provided herein, the compositionproduces one or more organic acids selected from the group consisting oflactate, butyrate, isobutyrate, propionate, acetate, isovalerate, andvalerate. In some embodiments of any of the embodiments provided herein,the composition further comprises one or more enzymes. In someembodiments, the one or more enzymes are selected from the groupconsisting of a phytase, a protease, an amylase, a xylanase, and abeta-glucanase. In some embodiments of any of the embodiments providedherein, the composition further comprises one or more essential oils. Insome embodiments of any of the embodiments provided herein, each strainis present at a concentration of at least about 1×10³ CFU/g feedadditive composition to at least about 1×10⁹ CFU/g (such as about 1×10¹⁵CFU/g) feed additive composition. In some embodiments of any of theembodiments provided herein, the composition inhibits at least onepathogen selected from avian pathogenic Salmonella sp., Escherichiacoli, Clostridium perfringens and Enterobacteriaceae in agastrointestinal tract of a bird having ingested an effective amount ofsaid direct fed microbial composition. In some embodiments of any of theembodiments provided herein, the composition is formulated for deliveryto an animal via waterline.

In other aspects, provided herein is a bacterial consortium comprising(a) a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.reuteri strain S1 deposited at Westerdijk Fungal Biodiversity Institute(WFDB) under number CBS 145921; and (b) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of a L. reuteri strain S2 deposited at WFDBunder number CBS 145922; and (c) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of a L. reuteri strain S3 deposited at WFDBunder number CBS 145923. In some embodiments, the consortium comprises(a) L. reuteri strain S1 (CBS 145921) or a live strain having all of theidentifying characteristics of L. reuteri strain S1 (CBS 145921); and(b) L. reuteri strain S2 (CBS 145922) or a live strain having all of theidentifying characteristics of L. reuteri strain S2 (CBS 145922); and(c) L. reuteri strain S3 (CBS 145923) or a live strain having all of theidentifying characteristics of L. reuteri strain S3 (CBS 145923) either(A) alone; or (B) in combination with a culture supernatant derived fromeach of these strains. In some embodiments, the consortium comprises a)the 16S ribosomal RNA sequence of L. reuteri strain S1 comprises thenucleotide sequence of SEQ ID NO:7; and (b) the 16S ribosomal RNAsequence of L. reuteri strain S2 comprises the nucleotide sequence ofSEQ ID NO:8; and (c) the 16S ribosomal RNA sequence of L. reuteri strainS3 comprises the nucleotide sequence of SEQ ID NO:9. In some embodimentsof any of the embodiments disclosed herein, one or more bacterialstrains further comprise one or more inactivated or deletedAntimicrobial resistance (AMR) genes. In some embodiments of any of theembodiments disclosed herein, the consortium produces one or moreorganic acids selected from the group consisting of lactate, butyrate,isobutyrate, propionate, acetate, isovalerate, and valerate. In someembodiments of any of the embodiments disclosed herein, each strain ispresent at a concentration of at least about 1×10³ CFU/animal/day to atleast about 1×10¹⁵ CFU/animal/day in the consortium. hi some embodimentsof any of the embodiments disclosed herein, the consortium inhibits atleast one pathogen selected from avian pathogenic Salmonella sp.,Escherichia coli, Clostridium perfringens and Enterobacteriaceae in agastrointestinal tract of a bird having ingested an effective amount ofsaid direct fed microbial composition.

In further aspects, provided herein is a bacterial consortium comprising(a) a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.salivarius strain 112 deposited at WFDB under number CBS 145919; and (b)a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.gallinarum strain H1 deposited at WFDB under number CBS 145918; and(c)(i) a bacterial strain having a 16S ribosomal RNA sequence displayingat least 97.0% sequence similarity to a 16S ribosomal RNA sequence of anL. agilis strain H3 comprising SEQ ID NO:1; or (ii) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of an L. reuteri strain A2deposited at WFDB under number CBS 145924. In some embodiments, theconsortium comprises (a) L. salivarius strain H2 (CBS 145919) or a livestrain having all of the identifying characteristics of L. salivariusstrain H2 (CBS 145919); and (b) L. gallinarum strain H1 (CBS 145918) ora live strain having all of the identifying characteristics of L.gallinarum strain H1 (CBS 145918); and (c) L. reuteri strain A2 (CBS145924) or a live strain having all of the identifying characteristicsof L. reuteri strain A2 (CBS 145924) either (A) alone; or (B) incombination with a culture supernatant derived from each of thesestrains. In some embodiments of any of the embodiments disclosed herein,one or more bacterial strains further comprise one or more inactivatedor deleted Antimicrobial resistance (AMR) genes. In some embodiments ofany of the embodiments disclosed herein, the consortium produces one ormore organic acids selected from the group consisting of lactate,butyrate, isobutyrate, propionate, acetate, isovalerate, and valerate.In some embodiments of any of the embodiments disclosed herein, eachstrain is present at a concentration of at least about 1×10³CFU/animal/day to at least about 1×10¹⁵ CFU/animal/day in theconsortium. In some embodiments of any of the embodiments disclosedherein, the consortium inhibits at least one pathogen selected fromavian pathogenic Salmonella sp., Escherichia coli, Clostridiumperfringens and Enterobacteriaceae in a gastrointestinal tract of a birdhaving ingested an effective amount of said direct fed microbialcomposition.

In still additional aspects, provided herein is a bacterial consortiumcomprising (a) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of an L. salivarius strain A1 comprising SEQ ID NO:2; and (b) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.agilis strain A3 comprising SEQ ID NO:3; and (c) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of an L. reuteri strain A2deposited at WFDB under number CBS 145924 either (A) alone; or (B) incombination with a culture supernatant derived from each of thesestrains. In some embodiments, the consortium comprises L. reuteri strainA2 (CBS 145924) or a live strain having all of the identifyingcharacteristics of L. reuteri strain A2 (CBS 145924). In someembodiments of any of the embodiments disclosed herein, one or morebacterial strains further comprise one or more inactivated or deletedAntimicrobial resistance (AMR) genes. In some embodiments of any of theembodiments disclosed herein, the consortium produces one or moreorganic acids selected from the group consisting of lactate, butyrate,isobutyrate, propionate, acetate, isovalerate, and valerate. In someembodiments of any of the embodiments disclosed herein, each strain ispresent at a concentration of at least about 1×10³ CFU/animal/day to atleast about 1×10¹⁵ CFU/animal/day in the consortium. In some embodimentsof any of the embodiments disclosed herein, the consortium inhibits atleast one pathogen selected from avian pathogenic Salmonella sp.,Escherichia coli, Clostridium perfringens and Enterobacteriaceae in agastrointestinal tract of a bird having ingested an effective amount ofsaid direct fed microbial composition.

In another aspect, provided herein is a bacterial consortium comprising(a) a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.salivarius strain D2 comprising SEQ ID NO:4; (b) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of an L. agilis strain D1comprising SEQ ID NO:5; and (c) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of an L. crispatus strain D3 comprising SEQID NO:6 either (A) alone; or (B) in combination with a culturesupernatant derived from each of these strains. In some embodiments ofany of the embodiments disclosed herein, one or more bacterial strainsfurther comprise one or more inactivated or deleted Antimicrobialresistance (AMR) genes. In some embodiments of any of the embodimentsdisclosed herein, the consortium produces one or more organic acidsselected from the group consisting of lactate, butyrate, isobutyrate,propionate, acetate, isovalerate, and valerate. In some embodiments ofany of the embodiments disclosed herein, each strain is present at aconcentration of at least about 1×10³ CFU/animal/day to at least about1×10¹⁵ CFU/animal/day in the consortium. In some embodiments of any ofthe embodiments disclosed herein, the consortium inhibits at least onepathogen selected from avian pathogenic Salmonella sp., Escherichiacoli, Clostridium perfringens and Enterobacteriaceae in agastrointestinal tract of a bird having ingested an effective amount ofsaid direct fed microbial composition.

In other aspects, provided herein is a premix comprising any of the feedadditive compositions disclosed herein or any of the bacterial consortiadisclosed herein and at least one mineral and/or at least one vitamin.

In additional aspects, provided herein is a feed comprising any of thefeed additive compositions disclosed herein or any of the premixesdisclosed herein or any of the bacterial consortia disclosed herein.

In yet other aspects, provided herein is a kit comprising a) any of thefeed additive compositions disclosed herein or any of the bacterialconsortia disclosed herein; and b) written instructions foradministration to an animal. In some embodiments, the kit furthercomprises one or more enzymes. In some embodiments, the one or moreenzymes are selected from the group consisting of a phytase, a protease,an amylase, a xylanase and a beta-glucanase.

In another aspect, provided herein is a method for improving one or moremetrics in an animal selected from the group consisting of increasedbodyweight gain, intestinal health status, decreased feed conversionratio (FCR), improved gut barrier integrity, reduced mortality, reducedpathogen infection, and reduced pathogen shedding in feces comprisingadministering an effective amount of any of the bacterial consortiadisclosed herein, or any of the feed additive compositions disclosedherein, any of the premixes disclosed herein, or any of the feedsdisclosed herein, thereby improving the one or more metrics in theanimal. In some embodiments, the feed additive composition increases oneor more of the lactate, acetate, isobutyrate, butyrate, isovalerate,and/or valerate content of the gastrointestinal tract of the animal. Insome embodiments of any of the embodiments provided herein, the pathogenis one or more of Clostridium perfringens, Campylobacter jejun,Enterobacteriaceae, a Salmonella sp., and/or Escherichia coli. In someembodiments of any of the embodiments provided herein, the methodfurther treats, prevents, or decreases incidence of necrotic enteritis.In some embodiments of any of the embodiments provided herein, theanimal is a domesticated bird. In some embodiments, the domesticatedbird is selected from the group consisting of chickens, turkeys, ducks,geese, quail, emus, ostriches, and pheasant. In some embodiments, thechicken is a broiler or a layer.

In still further aspects, provided herein is a method for preparing afeed additive composition or a bacterial consortium comprising combining(a) a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.reuteri strain S1 deposited at Westerdijk Fungal Biodiversity Institute(WFDB) under number CBS 145921; and (b)(i) a bacterial strain having a16S ribosomal RNA sequence displaying at least 97.0% sequence similarityto a 16S ribosomal RNA sequence of a L. reuteri strain S2 deposited atWFDB under number CBS 145922; and (ii) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of aL. reuteri strain S3 deposited at WFDBunder number CBS 145923. In some embodiments, (a) the L. reuteri strainS1 is a L. reuteri strain S1 (CBS 145921) or a live strain having all ofthe identifying characteristics of L. reuteri strain S1 (CBS 145921);and (b)(i) the L. reuteri strain S2 is aL. reuteri strain S2 (CBS145922) or a live strain having all of the identifying characteristicsof L. reuteri strain S2 (CBS 145922); and (ii) the L. reuteri strain S3is a L. reuteri strain S3 (CBS 145923) or a live strain having all ofthe identifying characteristics of L. reuteri strain S3 (CBS 145923)either (A) alone; or (B) in combination with a culture supernatantderived from each of these strains. In some embodiments of any of theembodiments disclosed herein, the method further comprises combining oneor more enzyme(s) with the feed additive composition. In someembodiments, the one or more enzymes are selected from the groupconsisting of a phytase, a protease, an amylase, a xylanase and abeta-glucanase. In some embodiments of any of the embodiments disclosedherein, at least about 1×10³ CFU/g feed additive composition to at leastabout 1×10⁹ CFU/g feed additive composition is combined to form the feedadditive composition. In some embodiments of any of the embodimentsdisclosed herein, the method further comprises packaging the feedadditive composition.

In another aspect, provided herein is a method for preparing a feedadditive composition or a bacterial consortium comprising combining (a)a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.salivarius strain H2 deposited at WFDB under number CBS 145919; and(b)(i) a bacterial strain having a 16S ribosomal RNA sequence displayingat least 97.0% sequence similarity to a 16S ribosomal RNA sequence of aL. gallinarum strain H1 deposited at WFDB under number CBS 145918; and(ii)(A) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of an L. agilis strain H3 comprising SEQ ID NO:1; or (B) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.reuteri strain A2 deposited at WFDB under number CBS 145924. In someembodiments, (a) the L. salivarius strain H2 is an L. salivarius strainH2 (CBS 145919) or a live strain having all of the identifyingcharacteristics of L. salivarius strain H2 (CBS 145919); and (b)(i) theL. gallinarum strain H1 is an L. gallinarum strain H1 (CBS 145918) or alive strain having all of the identifying characteristics of L.gallinarum strain H1 (CBS 145918); and (ii) the L. reuteri strain A2 isan L. reuteri strain A2 (CBS 145924) or a live strain having all of theidentifying characteristics of L. reuteri strain A2 (CBS 145924) either(A) alone; or (B) in combination with a culture supernatant derived fromeach of these strains. In some embodiments of any of the embodimentsdisclosed herein, the method further comprises combining one or moreenzyme(s) with the feed additive composition. In some embodiments, theone or more enzymes are selected from the group consisting of a phytase,a protease, an amylase, a xylanase and a beta-glucanase. In someembodiments of any of the embodiments disclosed herein, at least about1×10³ CFU/g feed additive composition to at least about 1×10⁹ CFU/g feedadditive composition is combined to form the feed additive composition.In some embodiments of any of the embodiments disclosed herein, themethod further comprises packaging the feed additive composition.

In other aspects, provided herein is a method for preparing a feedadditive composition or a bacterial consortium comprising combining (a)a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.salivarius strain A1 comprising SEQ ID NO:2; and (b)(i) a bacterialstrain having a 16S ribosomal RNA sequence displaying at least 97.0%sequence similarity to a 16S ribosomal RNA sequence of an L. agilisstrain A3 comprising SEQ ID NO:3; and (ii) a bacterial strain having a16S ribosomal RNA sequence displaying at least 97.0% sequence similarityto a 16S ribosomal RNA sequence of an L. reuteri strain A2 deposited atWFDB under number CBS 145924 either (A) alone: or (B) in combinationwith a culture supernatant derived from each of these strains. In someembodiments, the L. reuteri strain A2 is a L. reuteri strain A2 (CBS145924) or a live strain having all of the identifying characteristicsof L. reuteri strain A2 (CBS 145924). In some embodiments of any of theembodiments disclosed herein, the method further comprises combining oneor more enzyme(s) with the feed additive composition. In someembodiments, the one or more enzymes are selected from the groupconsisting of a phytase, a protease, an amylase, a xylanase and abeta-glucanase. In some embodiments of any of the embodiments disclosedherein, at least about 1×10³ CFU/g feed additive composition to at leastabout 1×10⁹ CFU/g feed additive composition is combined to form the feedadditive composition. In some embodiments of any of the embodimentsdisclosed herein, the method further comprises packaging the feedadditive composition.

In further aspects, provided herein is a method for preparing a feedadditive composition or a bacterial consortium comprising combining (a)a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.salivarius strain D2 comprising SEQ ID NO:4; (b)(i) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of an L. agilis strain D1comprising SEQ ID NO:5; and (ii) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of an L. crispatus strain D3 comprising SEQID NO:6 either (A) alone; or (B) in combination with a culturesupernatant derived from each of these strains. In some embodiments ofany of the embodiments disclosed herein, the method further comprisescombining one or more enzyme(s) with the feed additive composition. Insome embodiments, the one or more enzymes are selected from the groupconsisting of a phytase, a protease, an amylase, a xylanase and abeta-glucanase. In some embodiments of any of the embodiments disclosedherein, at least about 1×10³ CFU/g feed additive composition to at leastabout 1×10⁹ CFU/g feed additive composition is combined to form the feedadditive composition. In some embodiments of any of the embodimentsdisclosed herein, the method further comprises packaging the feedadditive composition.

In other aspects, provided herein is a method for preparing a premixcomprising combining any of the feed additive compositions disclosedherein with at least one mineral and/or at least one vitamin. In someembodiments, the method further comprises packaging the premix.

Each of the aspects and embodiments described herein are capable ofbeing used together, unless excluded either explicitly or clearly fromthe context of the embodiment or aspect.

Throughout this specification, various patents, patent applications andother types of publications (e.g., journal articles, electronic databaseentries, etc.) are referenced. The disclosure of all patents, patentapplications, and other publications cited herein are herebyincorporated by reference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph representing cumulative mortality data over timefor all houses in large-scale animal study.

DETAILED DESCRIPTION

A variety of microbial species have been shown to have certain degreesof efficacy against gut pathogens either in vitro or in vivo. Asdescribed in more detail herein, the inventors have surprisinglydiscovered that administering specific species of organic acid (such as,lactic acid)—producing microbes to animals (such as domesticated birds,for example, chickens) can improve performance on one or more metricsthat include increased bodyweight gain, intestinal health status,decreased feed conversion ratio (FCR), improved gut barrier integrity,reduced mortality, reduced pathogen infection (such as, but not limitedto, infection by Clostridium perfringens), and reduced pathogen sheddingin feces. Without being bound to theory, it is believed that metabolitesof organic acid-producing bacteria play an important role in theprevention of intestinal inflammation and in the maintenance ofintestinal homeostasis. While many bacterial species have the capabilityto produce organic acids, not all species can provide benefits toanimals when administered as a feed additive or as part of a feed.However, as will be described in the Examples section, administration ofparticular combinations of microbials was discovered to be surprisinglyeffective in the prevention and/or treatment of gut pathogenesis inanimals as well as for maintenance of overall health.

I. Definitions

“Organic acids,” as used herein, refers to an organic compound withacidic properties. In some non-limiting embodiments, organic acidcompounds are selected from the group consisting of lactic acid(2-hydroxypropionic acid), succinic acid, furandicarboxylic acid,fumaric acid, maleic acid, citric acid, glutamic acid, aspartic acid,acrylic acid, oxalic acid, and glucanic acid. Other non-limiting organicacids include formic acid (methanoic acid), acetic acid (ethanoic acid),propionic acid (propanoic acid), butanoic acid (butyric acid),isobutyric acid (2-methylpropanoic acid), valeric acid (pentanoic acid),and isovaleric acid (3-methylbutanoic acid). Inclusive in thisdefinition of organic acids are also the conjugate bases of organicacids including, for example, lactate, glutamate, fumarate, malate,formate, acetate, propionate, butyrate, isobutyrate, valerate,isovalerate etc.

As used herein, “microorganism” or “microbe” refers to a bacterium, afungus, a virus, a protozoan, and other microbes or microscopicorganisms.

As used here in the term “direct fed microbial” refers to a compositionfor consumption by animals (i.e. as an or as a component of animal feed)that contains viable microorganisms, i.e. microorganisms that arecapable of living and reproducing. See, for example, U.S. Pat. No.8,420,074. A direct fed microbial may comprise one or more (such as anyof 1, 2, 3, 4, 5, or 6 or more) of any of the microbial strainsdescribed herein.

A bacterial “strain” as used herein refers to a bacterium which remainsgenetically unchanged when grown or multiplied. The multiplicity ofidentical bacteria is included.

By “at least one strain,” is meant a single strain but also mixtures ofstrains comprising at least two strains of microorganisms. By “a mixtureof at least two strains,” is meant a mixture of two, three, fora, five,six or even more strains. In some embodiments of a mixture of strains,the proportions can vary from 1% to 99%. When a mixture comprises morethan two strains, the strains can be present in substantially equalproportions in the mixture or in different proportions.

For purposes of this disclosure, a “biologically pure strain” means astrain containing no other bacterial strains in quantities sufficient tointerfere with replication of the strain or to be detectable by normalbacteriological techniques. “Isolated,” when used in connection with theorganisms and cultures described herein, includes not only abiologically pure strain, but also any culture of organisms which isgrown or maintained other than as it is found in nature. In someembodiments, the strains are mutants, variants, or derivatives ofstrains A1, A2, A3, D1, D2, D3, H1, H2, H3, S1, S2, and S3 that alsoprovide benefits comparable to that provided by A1, A2, A3, D1, D2, D3,H1, H2, H3, S1, S2, and S3. In some embodiments, the strains are strainshaving all of the identifying characteristics of strains A1, A2, A3, D1,D2, D3, H1, H2, H3, S1, S2, and S3. Further, each individual strain (A1,A2, A3, D1, D2, D3, H1, H2, H3, S1, S2, and S3) or any combination ofthese strains can also provide one or more of the benefits describedherein. It will also be clear that addition of other microbial strains,carriers, additives, enzymes, yeast, or the like will also provide oneor more benefits or improvement of one or more metrics in an animal andwill not constitute a substantially different DFM.

The term “16S rRNA” or “16S ribosomal RNA” means the rRNA constitutingthe small subunit of prokaryotic ribosomes. In bacteria, this sequencecan be used to identify and characterize operational taxonomic units.

The term “sequence identity” or “sequence similarity” as used herein,means that two polynucleotide sequences, a candidate sequence and areference sequence, are identical (i.e. 100% sequence identity) orsimilar (i.e. on a nucleotide-by-nucleotide basis) over the length ofthe candidate sequence. In comparing a candidate sequence to a referencesequence, the candidate sequence may comprise additions or deletions(i.e. gaps) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. Optimal alignment of sequences for determining sequenceidentity may be conducted using the any number of publicly availablelocal alignment algorithms known in the art such as ALIGN or Megalign(DNASTAR), or by inspection.

The term “percent (%) sequence identity” or “percent (%) sequencesimilarity,” as used herein with respect to a reference sequence isdefined as the percentage of nucleotide residues in a candidate sequencethat are identical to the residues in the reference polynucleotidesequence after optimal alignment of the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity.

As used herein, “prevent,” “preventing,” “prevention” and grammaticalvariations thereof refers to a method of partially or completelydelaying or precluding the onset or recurrence of a disorder orcondition (such as necrotic enteritis) and/or one or more of itsattendant symptoms or barring an animal from acquiring or reacquiring adisorder or condition or reducing an animal's risk of acquiring orreacquiring a disorder or condition or one or more of its attendantsymptoms.

As used herein, the term “reducing” in relation to a particular trait,characteristic, feature, biological process, or phenomena refers to adecrease in the particular trait, characteristic, feature, biologicalprocess, or phenomena. The trait, characteristic, feature, biologicalprocess, or phenomena can be decreased by 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% orgreater than 100%.

The term “poultry,” as used herein, means domesticated birds kept byhumans for their eggs, their meat or their feathers. These birds aremost typically members of the superorder Galloanserae, especially theorder Galliformes which includes, without limitation, chickens, quails,ducks, geese, emus, ostriches, pheasant, and turkeys.

As used herein “administer” or “administering” is meant the action ofintroducing one or more microbial strain, an exogenous feed enzymeand/or a strain and an exogenous feed enzyme to an animal, such as byfeeding or by gavage.

As used herein, “effective amount” means a quantity of DFM and/orexogenous enzymes to improve one or more metrics in an animal.Improvement in one or more metrics of an animal (such as, withoutlimitation, any of increased bodyweight gain, intestinal health status,decreased feed conversion ratio (FCR), improved gut barrier integrity,reduced mortality, reduced pathogen infection, and reduced pathogenshedding in feces) can be measured as described herein or by othermethods known in the art. An effective amount can be administered to theanimal by providing ad libitum access to feed containing the DFM andexogenous enzymes. The DFM and exogenous enzymes can also beadministered in one or more doses.

The term “intestinal health status” refers to the status of the gut wallstructure and morphology which can be affected by, for example,infectious agents or a non-infectious cause, such as a suboptimalformulated diet. “Gut wall structure and morphology” or “gut barrierintegrity” can refer to, without limitation, epithelial damage andepithelial permeability which is characterized by a shortening of villi,a lengthening of crypts and an infiltration of inflammatory cells (suchas, without limitation. CD3+cells). The latter damage and inflammationmarkers can also be associated with a “severe” macroscopic appearance ofthe gut—compared to a “normal” appearance—when evaluated using a scoringsystem such as the one described by Teirlynck et al. (2011).

As used herein, the tern “feed” is used synonymously herein with“feedstuff” Feed broadly refers to a material, liquid or solid, that isused for nourishing an animal, and for sustaining normal or acceleratedgrowth of an animal including newborns or young and developing animals.The term includes a compound, preparation, mixture, or compositionsuitable for intake by an animal (such as, e.g., for poultry such asquail, ducks, turkeys, and chickens). In some embodiments, a feed orfeed composition comprises a basal food composition and one or more feedadditives or feed additive compositions. The term “feed additive” asused herein refers to components included for purposes of fortifyingbasic feed with additional components to promote feed intake, treat orprevent disease, or alter metabolism. Feed additives include pre-mixes.In other embodiments, a feed additive refers to a composition thatsupplements a feed but is not necessarily a component of the feed orfood. For example, in one embodiment, the feed additive compositionsupplements a feed via delivery through a fluid (such as water) that isadministered to the animal separately from the feed or food (forexample, via a waterline or water distribution system).

A “premix,” as referred to herein, may be a composition composed ofmicro-ingredients such as, but not limited to, one or more of vitamins,minerals, chemical preservatives, antibiotics, fermentation products,and other essential ingredients. Premixes are usually compositionssuitable for blending into commercial rations.

As used herein, “improving one or more metrics in an animal” refers toimprovements on measurements relevant to the growth and/or health of ananimal (such as a domesticated bird, for example, a chicken), measuredby one or more of the following parameters: average daily weight gain(ADG), overall weight, mortality, feed conversion (which includes bothfeed:gain and gain:feed), feed intake, intestinal health status,decreased feed conversion ratio (FCR), improved gut banter integrity,reduced mortality, reduced pathogen infection, and reduced pathogenshedding in feces. “An improvement in a metric” or “improved metric” asused herein, refers to an improvement in at least one of the parameterslisted under the metrics in an animal definition.

As used herein, the term “feed conversion ratio” (FCR) refers to theamount of feed fed to an animal to increase the weight of the animal bya specified amount. An improved feed conversion ratio means a lower feedconversion ratio. By “lower feed conversion ratio” or “improved feedconversion ratio” it is meant that the use of a feed additivecomposition in feed results in a lower amount of feed being required tobe fed to an animal to increase the weight of the animal by a specifiedamount compared to the amount of feed required to increase the weight ofthe animal by the same amount when the feed does not comprise said feedadditive composition.

The phrase “antibiotic resistance genes” or “antimicrobial resistance(AMR) genes,” as used interchangeably herein, refers to genes thatconfer resistance to antibiotics, for example by coding for enzymeswhich destroy it, by coding for surface proteins which prevent it fromentering a microorganism, actively exports it, or by being a mutant formof the antibiotic's target so that it can ignore it. Examples of AMRgenes may be found on the ARDB—Antibiotic Resistance Genes Database(Center for Bioinfonnatics and Computational Biology, University ofMaryland, College Park, Md. 20742; gene.https://ardb.cbcb.umd.edu/).Non-limiting examples of AMR genes include but are not limited toextended-spectrum beta lactamse (ESBL) genes, methicillin resistancegene, CTX-M-15; ndm-1,2,5,6, a vancomycin resistance, InuC (anucleotidyltrausferase), vatE, (an acetyltransferase), or tetW In someembodiments, one or more AMR genes can be associated with a mobilegenetic element (such as a transposon) near the gene (such as within anyof about 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or closer to the gene, including distances falling in between anyof these values). In other embodiments, one or more AMR genes are notassociated with a mobile genetic element, such as a transposon.

As used herein “mobile genetic element” is meant to include any type ofnucleic acid molecule that is capable of movement within a genome orfrom one genome to another. For example, these can include, withoutlimitation, transposons or transposable elements (includingretrotransposons, DNA transposons, and insertion sequences); plasmids;bacteriophage elements (including Mu; and group II introns).

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number can be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number. For example,in connection with a numerical value, the term “about” refers to a rangeof −10% to +10% of the numerical value, unless the term is otherwisespecifically defined in context.

As used herein, the singular terms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elementsor use of a “negative” limitation.

It is also noted that the tenu “consisting essentially of,” as usedherein refers to a composition wherein the component(s) after the termis in the presence of other known component(s) in a total amount that isless than 30% by weight of the total composition and do not contributeto or interferes with the actions or activities of the component(s).

It is further noted that the term “comprising,” as used herein, meansincluding, but not limited to, the component(s) after the term“comprising.” The component(s) after the tern “comprising” are requiredor mandatory, but the composition comprising the component(s) canfurther include other non-mandatory or optional component(s).

It is also noted that the term “consisting of,” as used herein, meansincluding, and limited to, the component(s) after the term “consistingof.” The component(s) after the term “consisting of” are thereforerequired or mandatory, and no other component(s) are present in thecomposition.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains.

Other definitions of terms may appear throughout the specification.

II. Compositions A. Strains

Direct fed microbials (DFMs) refer to the feeding of beneficial microbesto animals, such as domestic birds, when they are under periods ofstress (disease, ration changes, environmental or production challenges)or as a part of a daily nutritional regimen to prevent disease andfacilitate nutrient usage dining digestion. Probiotics is another termfor this category of feed additives. Probiotics or DFMs have been shownto improve animal performance in controlled studies. In someembodiments, DFMs include both direct fed bacteria and/or yeast-basedproducts and, in particular embodiments, include viable microorganisms.The term “viable microorganism” means a microorganism which ismetabolically active or able to differentiate.

In one embodiment, the DFM may be a spore forming bacterium and hencethe term DFM may refer to a composition that is comprised of or containsspores, e.g., bacterial spores. Therefore, in one embodiment the term“viable microorganism” as used herein may include microbial spores, suchas endospores or conidia. In another embodiment, the DFM in the feedadditive composition according to the present invention is not comprisedof or does not contain microbial spores, e.g. endospores or conidia(i.e., the DFM is non-spore forming).

The strains provided herein include Lactobacillus reuteri strain S1, L.reuteri strain S2, L. reuteri strain S3, L. gallinarum strain H1, L.salivarius strain H2, L. agilis strain H3, L. salivarius strain A1, L.reuteri strain A2, L. reuteri strain A3, L. agilis strain D1, L.salivarius strain D2, and L. crispatus strain D3, which are alsoreferred to herein as S1, S2, S3, H1, H2, H3, A1, A2, A3, D1, D2, andD3, respectively.

L. reuteri strain S1, L. reuteri strain S2, L. reuteri strain S3, L.reuteri strain A2, L. gallinarum strain H1, L. salivarius strain H2, andL. agilis strain H3 were deposited on Jul. 24, 2019 at the WesterdijkFungal Biodiversity Institute (WFDB), Uppsalalaan 8, 3584 CT, Utrecht,The Netherlands and given accession numbers CBS 145921, CBS 145922, CBS145923, CBS 145924, CBS145918, CBS145919, and CBS 145920, respectively.The deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. One or more strain provided herein can beused as a direct-fed microbial (DFM).

The DFM strains disclosed herein are primarily found in the genusLactobacillus. As of March 2020, Lactobacilli comprised 261 species thatare extremely diverse at phenotypic, ecological and genotypic. Givenrecent advances in whole genome sequencing and comparative genomics, thegenus Lactobacillus was recently divided into 25 separate genera withstrains belonging to previously designated Lactobacilli species beingtransferred to new species and/or genera (see Zheng et al., 2020, Int. JSvst. Evol. Microbiol. 70:2782-2858; Pot et al., Trends in Food Science& Technology 94 (2019) 105-113; and Koutsoumanis et al., 2020, EFSAJournal, 18(7):6174 the disclosures of each of which are incorporated byreference herein). For purposes of the instant disclosure, the previousclassification of Lactobacillus species will continue to be employed.However, in some embodiments Lactobacillus agilis is also classified asas Ligilactobacillus agilis. In other embodiments, Lactobacillussalivarius is also classified as Ligilactobacillus salivarius. Infurther embodiments, Lactobacillus reuteri is also classified asLimosilactobacillus reuteri.

DFM compositions can include those that contain one or more strains(such as any of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) ofLactobacillus reuteri; and/or L. salivarius. L. reuteri is agram-positive bacterium that naturally inhabits the gut of mammals andbirds. First described in the early 1980s, some strains of L. reuteriare used as probiotics. Some L. reuteri can produce a novelbroad-spectrum antibiotic substance known as reuterin. Lactobacillussalivarius is a probiotic bacteria species that has been found to livein the gastrointestinal tract and exert a range of therapeuticproperties including suppression of pathogenic bacteria. The DFMcomposition can further include those that contain one or more strains(such as any of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) of L.agilis, L. crispatus, and/or L. gallinarum.

The DFM composition can also include only strains of L. reuteri (such as1, 2, 3, 4, 5, 6, 7, or 8 strains of L. reuteri without any othermicrobials present). For example, the DFM composition can include one ormore of L. reuteri strains S1, S2, and/or S3 or one or more microbe(s)having a 16S ribosomal RNA sequence displaying at least about 97.0%sequence similarity (such as any of about 97%, 97.5%. 98%, 98.5%, 99%,99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequence ofone or more of L. reuteri strain S1 (SEQ ID NO:7), S2 (SEQ ID NO:8),and/or S3 (SEQ ID NO:9). In some embodiments, the DFM compositionincludes only L. reuteri strain S1, S2, or S3. In another embodiment,DFM composition includes L. reuteri strains S1 and S2, L. reuteristrains S1 and S3; L. reuteri strains S2 and S3; or L. reuteri strainsS1, S2, and S3. Additionally, when cultured together, one or more L.reuteri strains S1, S2, and/or S3 have one or more physiological ormetabolic properties that individually cultured L. reuteri strains lack.These properties can include, without limitation, changes in the amountand/or type of organic acid produced, change in metabolic profile,and/or a change in the composition of media in which the bacteria arecultured together (such as lactate).

The DFM compositions provided herein can include one or more of L.reuteri strains S1, S2, and/or S3 (i.e. the compositions include theactual bacteria from these strains) and/or one or more culturesupernatants derived from the culturing of these strains (individuallyor in co-culture).

DFM compositions can additionally include those that contain one or moreof L. salivarius microbes, L. gallinarum microbes. L. agilis microbes,and/or L. reuteri microbes.

The DFM composition can include one or more of L. salivarius strain H2,L. gallinarum strain H1, L. reuteri strain A2, and/or L. agilis strainH3 or one or more microbe(s) having a 16S ribosomal RNA sequencedisplaying at least about 97.0% sequence similarity (such as any ofabout 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity)to a 16S ribosomal RNA sequence of one or more of L. salivarius strainH2 (SEQ ID NO:10), L. gallinarum strain H1 (SEQ ID NO:11), L. reuteristrain A2 (SEQ ID NO:12), and/or L. agilis strain H3 (SEQ ID NO:1). Insome embodiments, the DFM composition includes only L. salivarius strainH2, L. gallinarum strain H1, L. reuteri strain A2, or L. agilis strainH3. In another embodiment, the DFM composition includes L. salivariusstrain H2 and L. gallinarum strain H1; L. salivarius strain H2 and L.reuteri strain A2; L. salivarius strain H2 and L. agilis strain H3; L.gallinarum strain H1 and L. reuteri strain A2; L. gallinarum strain H1and L. agilis strain H3; L. reuteri strain A2 and L. agilis strain H3;L. salivarius strain H2, L. gallinarum strain H1, and L. reuteri strainA2 ; L. salivarius strain H2, L. reuteri strain A2, and L. agilis strainH3: L. gallinarum strain H1, L. reuteri strain A2, and L. agilis strainH3; L. salivarius strain H2, L. gallinarum strain H1, L. reuteri strainA2, and L. agilis strain H3; L. salivarius strain H2, L. gallinarumstrain H1, and L. reuteri strain A2; or L. salivarius strain H2, L.gallinarum strain H1, and L. agilis strain H3. Additionally, whencultured together, one or more L. salivarius strain H2, L. gallinarumstrain H1, L. reuteri strain A2, and/or L. agilis strain H3 (such as L.salivarius strain H2, L. gallinarum strain H1, and L. reuteri strain A2;or L. salivarius strain H2, L. gallinarum strain H1, and L. agilisstrain H3) have one or more physiological or metabolic properties thatindividually cultured strains lack. These properties can include,without limitation, changes in the amount and/or type of organic acidproduction (such as the production of lactic acid).

The DFM compositions provided herein can include one or more L.salivarius strain H2, L. gallinarum strain H1, L. reuteri strain A2,and/or L. agilis strain H3 (such as L. salivarius strain H2, L.gallinarum strain H1, and L. reuteri strain A2; or L. salivarius strainH2, L. gallinarum strain H1, and L. agilis strain H3) (i.e. thecompositions include the actual bacteria from these strains) and/or oneor more culture supernatants derived from the culturing of these strains(individually or in co-culture).

DFM compositions can additionally include those that contain one or moreof L. salivarius microbes, L. agilis microbes, and/or L. reuterimicrobes.

The DFM composition can include one or more of L. salivarius strain A1,L. reuteri strain A2, and/or L. agilis strain A3 or one or moremicrobe(s) having a 16S ribosomal RNA sequence displaying at least about97.0% sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%,99%, 99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequenceof one or more of L. salivarius strain Al (SEQ ID NO:9), L. reuteristrain A2 (SEQ ID NO:10), and/or L. agilis strain A3 (SEQ ID NO:11). Insome embodiments, the DFM composition includes only L. salivarius strainAl, L. reuteri strain A2, or L. agilis strain A3. In another embodiment,the DFM composition includes L. salivarius strain A1 and L. reuteristrain A2; L. salivarius strain A1 and L. agilis strain A3; L. reuteristrain A2 and L. agilis strain A3; L. salivarius strain A1, L. reuteristrain A2, and L. agilis strain A3. Additionally, when culturedtogether, one or more L. salivarius strain A1, L. reuteri strain A2,and/or L. agilis strain A3 have one or more physiological or metabolicproperties that individually cultured strains lack. These properties caninclude, without limitation, changes in the amount and/or type oforganic acid produced (such as the production of lactic acid) change inmetabolic profile, and/or a change in the composition of media in whichthe bacteria are cultured together.

The DFM compositions provided herein can include one or more of L.salivarius strain A1, L. reuteri strain A2, and/or L. agilis strain A3(i.e. the compositions include the actual bacteria from these strains)and/or one or more culture supernatants derived from the culturing ofthese strains (individually or in co-culture).

DFM compositions can additionally include those that contain one or moreof L. salivarius microbes, L. agilis microbes, and/or L. crispatusmicrobes.

The DFM composition can include one or more of L. agilis strain D1, L.salivarius strain D2, and/or L. crispatus strain D3 or one or moremicrobe(s) having a 16S ribosomal RNA sequence displaying at least about97.0% sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%,99%, 99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequenceof one or more of L. agilis strain D1 (SEQ ID NO:5), L. salivariusstrain D2 (SEQ ID NO:4), and/or L. crispatus strain D3 (SEQ ID NO:6). Insome embodiments, the DFM composition includes only L. agilis strain D1,L. salivarius strain D2, or L. crispatus strain D3. In anotherembodiment, the DFM composition includes L. agilis strain D1 and L.salivarius strain D2; L. agilis strain D1 and L. crispatus strain D3; L.salivarius strain D2 and L. crispatus strain D3; L. agilis strain D1, L.salivarius strain D2, and L. crispatus strain D3. Additionally, whencultured together, one or more L. agilis strain D1, L. salivarius strainD2, and/or L. crispatus strain D3 have one or more physiological ormetabolic properties that individually cultured strains lack. Theseproperties can include, without limitation, changes in the amount and/ortype of organic acid produced (such as the production of lactic acid)change in metabolic profile, and/or a change in the composition of mediain which the bacteria are cultured together.

The DFM compositions provided herein can include one or more L. agilisstrain D1, L. salivarius strain D2, and/or L. crispatus strain D3 (i.e.the compositions include the actual bacteria from these strains) and/orone or more culture supernatants derived from the culturing of thesestrains (individually or in co-culture).

In some embodiments, one or more of (such as any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or 11) the strains provided herein including L. reuteri strainS1 (CBS 145921), L. reuteri strain S2 (CBS 145922), L. reuteri strain S3(CBS 145923), L. gallinarum strain H1 (CBS145918), L. salivarius strainH2 (CBS 145919), L. reuteri strain A2 (CBS 145924), L. agilis strain H3,L. salivarius strain A1, L. agilis strain A3, L. agilis strain D1, L.salivarius strain D2, and L. crispatus strain D3 further comprise one ormore (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) inactivatedor deleted AMR genes.

B. Exogenous Enzymes

Supplemental enzymes can be used as additives to animal feed,particularly poultry and swine feeds, as a means to improve nutrientutilization and performance characteristics.

In one embodiment, the disclosure relates to a composition comprisingone or more DFM (such as DFMs containing any of the microbial strainsdisclosed herein) and one or more exogenous feed enzymes. In anotherembodiment, the disclosure relates to a composition comprising,consisting of, or consisting essentially of a multi-strain DFM (such asany of the multi-strain DFM compositions disclosed herein) and one ormore exogenous feed enzymes. In one embodiment, the exogenous feedenzymes include, but are not limited to, xylanase, amylase, phytase,beta-glucanase, and protease. In still another embodiment, thecomposition comprises a feed additive.

1. Xylanases

Xylanase is the name given to a class of enzymes that degrade the linearpolysaccharide β-1,4-xylan into xylose, thus breaking downhemicellulose, one of the major components of plant cell walls.Xylanases, e.g., endo-β-xylanases (EC 3.2.1.8) hydrolyze the xylanbackbone chain. In one embodiment, provided herein are compositionscomprising a multi-strain DFM (such as any of the multi-strain DFMcompositions disclosed herein) and one or more xylanase.

In one embodiment, the xylanase may be any commercially availablexylanase. Suitably the xylanase may be an endo-1,4-P-d-xylanase(classified as E.G. 3.2.1.8) or a 1,4β-xylosidase (classified as E.G.3.2.1.37). In one embodiment. the disclosure relates to a DFM incombination with an endoxylanase, e.g. an endo-1,4-P-d-xylanase, andanother enzyme. All E.C. enzyme classifications referred to hereinrelate to the classifications provided in EnzymeNomenclature—Recommendations (1992) of the nomenclature committee of theInternational Union of Biochemistry and Molecular Biology—ISBN0-12-226164-3, which is incorporated herein

In another embodiment, the xylanase may be a xylanase from Bacillus,Trichodermna, Therinomyces, Aspergillus and Penicillium. In stillanother embodiment, the xylanase may be the xylanase in Axtra XAP® orAvizyme 1502®. both commercially available products from Danisco A/S. Inone embodiment, the xylanase may be a mixture of two or more xylanases.In still another embodiment, the xylanase is an endo-1,4-β-xylanase or a1,4-β-xylosidase. In yet another embodiment, the xylanase is from anorganism selected from the group consisting of: Bacillus, Trichoderma,Thermomyces, Aspergillus, Penicillium, and Humicola. In yet anotherembodiment, the xylanase may be one or more of the xylanases or one ormore of the commercial products recited in Table 1.

TABLE 1 Representative commercial xylanases Representative examples ofcommercial xylanases. Commercial Name

Company Xylanase type Xylanase source Allzyme PT Alltechendo-1,4-β-xylanase Aspergillus Niger Amylofeed Andr{tilde over (e)}sPintaluba

endo-1,4-β-xylanase Aspergillus Niger

endo-1,4-β-xylanase Trichoderma reesei

endo-1,4-β-xylanase Trichoderma reesei Avizyme 1100 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1110 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1202 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1210 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1302 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme 1500 Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum Avizyme SX Daniscoendo-1,4-β-xylanase Trichoderma longibrachiatum

Danisco endo-1,4-β-xylanase Trichoderma longibrachiatum Belfeed MP

Beldem endo-1,4-β-xylanase Bacillus subtilis Biofeed Plus DSMendo-1,4-β-xylanase Humicola insolens Danisco Danisco Animalendo-1,4-β-xylanase Trichoderma reesei Glycosidase Nutrition

Da

co Danisco endo-1,4-β-xylanase Trichoderma reesei Xylanase

AB Vista endo-1,4-β-xylanase Trichoderma reesei Endofeed ® Andr{tildeover (e)}s Pintaluba endo-1,4-β-xylanase Aspergillus Niger

Lyven endo-1,4-β-xylanase Trichoderma longibrachiatum Grindazym GPDanisco endo-1,4-β-xylanase Aspergillus Niger Grindazym GV Daniscoendo-1,4-β-xylanase Aspergillus Niger Hostazym X

endo-1,4-β-xylanase Trichoderma longibrachiatum Kemzyme Plus Keminendo-1,4-β-xylanase Trichoderma viride Dry Kemzyme Plus Keminendo-1,4-β-xylanase Trichoderma viride Liquid Kemzyme W dry Keminendo-1,4-β-xylanase Trichoderma viride Kemzyme W dry Keminendo-1,4-β-xylanase Trichoderma viride liquid Natugrain BASFendo-1,4-β-xylanase Trichoderma longibrachiatum Natugrain TS BASFendo-1,4-β-xylanase Aspergillus Niger Plus Natugrain Wheat BASFendo-1,4-β-xylanase Aspergillus Niger Natugrain ® TSL BASFendo-1,4-β-xylanase Aspergillus Niger Natuzyme Bioprotonendo-1,4-β-xylanase Trichoderma longibrachiatum Trichoderma reeseiPorzyme 8100 Danisco endo-1,4-β-xylanase Trichoderma longibrachiatumPorzyme 8300 Danisco endo-1,4-β-xylanase Trichoderma longibrachiatumPorzyme 9102 Danisco endo-1,4-β-xylanase Trichoderma longibrachiatumPorzyme Danisco endo-1,4-β-xylanase Trichoderma longibrachiatum

Porzyme

Danisco endo-1,4-β-xylanase Trichoderma longibrachiatum Ronozyme AX DSMendo-1,4-β-xylanase Thermomyces lanuginosus gene expressed inAspergillus Aspergillus oryzae Ronozyme WX DSM Novozymesendo-1,4-β-xylanase Thermomyces lanuginosus gene expressed inAspergillus Aspergillus oryzae

endo-1,4-β-xylanase Penicillium funiculosum

DSM Novozymes endo-1,4-β-xylanase Trichoderma longibrachiatum

endo-1,4-β-xylanase Trichoderma longibrachiatum Xylanase

endo-1,4-β-xylanase Trichoderma longibrachiatum

indicates data missing or illegible when filed

In one embodiment, the disclosure relates to a composition comprising amulti-strain DFM (such as any of the multi-strain DFM compositionsdisclosed herein) and xylanase. In one embodiment, the compositioncomprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350,350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750,and greater than 750 xylanase units/g of composition.

In one embodiment, the composition comprises 500-1000, 1000-1500,1500-2000, 2000- 2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500,4500-5000, 5000-5500, 5500-6000, 6000- 6500, 6500-7000, 7000-7500.7500-8000, and greater than 8000 xylanase units/g composition.

It will be understood that one xylanase unit (XU) is the amount ofenzyme that releases 0.5 μmol of reducing sugar equivalents (as xyloseby the Dinitrosalicylic acid (DNS) assay-reducing sugar method) from anoat-spelt-xylan substrate per min at pH 5.3 and 50° C. (Bailey, et al..Journal of Biotechnology, Volume 23, (3), May 1992, 257-270).

2. Amylases

Amylase is a class of enzymes capable of hydrolysing starch toshorter-chain oligosaccharides, such as maltose. The glucose moiety canthen be more easily transferred from maltose to a monoglyceride orglycosyhnonoglyceride than from the original starch molecule. The tem'amylase includes α-amylases (E.G. 3.2.1.1), G4-forming amylases (E.G.3.2.1.60), β-amylases (E.G. 3.2.1.2) and γ-amylases (E.C. 3.2.1.3).Amylases may be of bacterial or fungal origin, or chemically modified orprotein engineered mutants. In one embodiment, provided herein arecompositions comprising a multi-strain DFM (such as any of themulti-strain DFM compositions disclosed herein) and one or more amylase.

In one embodiment, the amylase may be a mixture of two or more amylases.In another embodiment, the amylase may be an amylase, e.g. an α-amylase,from Bacillus licheniformis and an amylase, e.g. an α-amylase, fromBacillus amyloliquefaciens. In one embodiment, the α- amylase may be theα-amylase in Axtra XAP® or Avizyme 1502®, both commercially availableproducts from Danisco A/S. In yet another embodiment, the amylase may bea pepsin resistant α-amylase, such as a pepsin resistant Trichoderma(such as Trichoderma reesei) alpha amylase. A suitably pepsin resistanta-amylase is taught in UK application number 101 1513.7 (which isincorporated herein by reference) and PCT/IB2011/053018 (which isincorporated herein by reference).

In one embodiment, the amylase for use in the present invention may beone or more of the amylases in one or more of the commercial productsrecited in Table 2.

TABLE 2 Representative commercial amylases Representative examples ofcommercial #Z,899; Commercial product

Company Amylase type Amylase source Amylofeed Andr{tilde over (e)}salpha amylase Aspergillus oryzae Pintaluba

Avizyme 1500 Danisco alpha amylase Bacillus amyloliquefaciens Avizyme1505 Danisco alpha amylase Bacillus amyloliquefaciens Kemzyme Plus DryKemin alpha-amylase Bacillus amyloliquefaciens Kemzyme Plus Keminalpha-amylase Bacillus amyloliquefaciens Liquid Kemzyme W dry Keminalpha-amylase Bacillus amyloliquefaciens Kemzyme W Kemin alpha-amylaseBacillus amyloliquefaciens liquid Natuzyme Bioproton alpha-amylaseTrichoderma longibrachiatum Trichoderma reesei Porzyme

Danisco alpha-amylase Bacillus amyloliquefaciens Porzyme

Danisco alpha-amylase Bacillus amyloliquefaciens Ronozyme A DSMalpha-amylase Bacillus amyloliquefaciens Novozymes Ronozyme AX DSMalpha-amylase Bacillus amyloliquefaciens Ronozyme ® DSM alpha-amylaseBacillus amyloliquefaciens

Novozymes expressed in Bacillus licheniformis

indicates data missing or illegible when filed

It will be understood that one amylase unit (AU) is the amount of enzymethat releases 1 mmol of glucosidic linkages from a water insolublecross-linked starch polymer substrate per min at pH 6.5 and 37° C. (thismay be referred to herein as the assay for determining 1 AU).

In one embodiment, disclosure relates to a composition comprising amulti-strain DFM (such as any of the multi-strain DFM compositionsdisclosed herein) and amylase. In one embodiment, disclosure relates toa composition comprising a multi-strain DFM, xylanase and amylase. Inone embodiment, the composition comprises 10-50, 50-100, 100-150,150-200, 200- 250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550,550-600, 600-650, 650-700, 700- 750, and greater than 750 amylaseunits/g composition.

In one embodiment, the composition comprises 500-1000, 1000-1500,1500-2000, 2000- 2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500,4500-5000, 5000-5500, 5500-6000, 6000- 6500, 6500-7000, 7000-7500,7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000,11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than15000 amylase units/g composition.

3. Proteases

The term protease as used herein is synonymous with peptidase orproteinase. The protease may be a subtilisin (E.G. 3.4.21.62) or abacillolysin (E.G. 3.4.24.28) or an alkaline serine protease (E.G.3.4.21.x) or a keratinase (E.G. 3.4.X.X). In one embodiment, theprotease is a subtilisin. Suitable proteases include those of animal,vegetable or microbial origin. Chemically modified or protein engineeredmutants are also suitable. The protease may be a serine protease or ametalloprotease. e.g., an alkaline microbial protease or a trypsin-likeprotease. In one embodiment, provided herein are compositions comprisinga multi-strain DFM (such as any of the multi-strain DFM compositionsdisclosed herein) and one or more protease.

Examples of alkaline proteases are subtilisins, especially those derivedfrom Bacillus sp., e.g., subtilisin Novo, subtilisin Carlsberg,subtilisin 309 (see, e.g., U.S. Pat. No. 6,287,841), subtilisin 147, andsubtilisin 168 (see, e.g., WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin), and Fusariumproteases (see, e.g., WO 89/06270 and WO 94/25583). Examples of usefulproteases also include but are not limited to the variants described inWO 92/19729 and WO 98/20115.

In another embodiment, the protease may be one or more of the proteasesin one or more of the commercial products recited in Table 3.

TABLE 3 Representative commercial proteases Representative examples ofcommercial proteases Commercial product ® Company Protease type Proteasesource Avizyme 1100 Danisco A/S Subtilisin Bacillus subtilis Avizyme1202 Danisco A/S Subtilisin Bacillus subtilis Avizyme 1302 Danisco A/SSubtilisin Bacillus subtilis Avizyme 1500 Danisco A/S SubtilisinBacillus subtilis Avizyme 1505 Danisco A/S Subtilisin Bacillus subtilisKemzyme Plus Dry Kemin Bacillolysin Bacillus amyloliquefaciens Kemzyme Wdry Kemin Bacillolysin Bacillus amyloliquefaciens Natuzyme BioprotonProtease Trichoderma longibrachiatum Trichoderma reesei Porzyme 8300Danisco Subtilisin Bacillus subtilis Ronozyme ProAct DSM NovozymesAlkaline

serine protease gene expressed in Bacillus licheniformis Versazyme

Novus Keratinase Bacillus licheniformis

indicates data missing or illegible when filed

In one embodiment, the protease is selected from the group consisting ofsubtilisin, a bacillolysin, an alkine serine protease, a kerativase, anda Nocardiopsis protease.

It will be understood that one protease unit (PU) is the amount ofenzyme that liberates from the substrate (0.6% casein solution) onemicrogram of phenolic compound (expressed as tyrosine equivalents) inone minute at pH 7.5 (40 mM Na₂PO₄/lactic acid buffer) and 40° C. Thismay be referred to as the assay for determining 1 PU.

In one embodiment, disclosure relates to a composition comprising amulti-strain DFM (such as any of the multi-strain DFM compositionsdisclosed herein) and a protease. In another embodiment, disclosurerelates to a composition comprising a multi-strain DFM (such as any ofthe multi-strain DFM compositions disclosed herein) and a xylanase and aprotease. In still another embodiment, the disclosure relates to acomposition comprising a multi-strain DFM (such as any of themulti-strain DFM compositions disclosed herein) and an amylase and aprotease. In yet another embodiment, the disclosure relates to acomposition comprising a multi-strain DFM (such as any of themulti-strain DFM compositions disclosed herein) and a xylanase, anamylase and a protease.

In one embodiment, the composition comprises 10-50, 50-100, 100-150,150-200, 200- 250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550,550-600, 600-650, 650-700, 700- 750, and greater than 750 proteaseunits/g composition.

In one embodiment, the composition comprises 500-1000, 1000-1500,1500-2000, 2000- 2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500,4500-5000, 5000-5500, 5500-6000, 6000- 6500, 6500-7000, 7000-7500,7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000,11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than15000 protease units/g composition.

4. Phytases

In one embodiment, provided herein are compositions comprising amulti-strain DFM (such as any of the multi-strain DFM compositionsdisclosed herein) and one or more phytase. The phytase for use in thepresent invention may be classified a 6-phytase (classified as E.C.3.1.3.26) or a 3-phytase (classified as E.C. 3.1.3.8). In oneembodiment, the phytase for use in the present invention may be one ormore of the phytases in one or more of the commercial products below inTable 4:

TABLE 4 Representative commercial phytases Commercial product ® CompanyPhytase type Phytase source

ABVista 3-phytase Trichoderma reesei

ABVista 6-phytase E. coli gene expressed in Trichoderma reesei NatuphosBASF 3-phytase Aspergillus Niger Natuzyme Bioproton phytase (typeTrichoderma longibrachiatum not specified) OPTIPHOS ® Huvepharma AD6-phytase E. coli gene expressed in Pichia pastoris Phytase

DSM 3-phytase A consensus gene expressed Hansenula polymorpha Phyzyme XPDanisco 6-phytase E. coli gene expressed in Schizosaccharomyces pombeQuantum

ABVista 6-phytase E. coli gene expressed in 5000L Pichia pastoris orTrichoderma longibrachiatum Ronozyme Hi-Plus DSM

Novozymes 6-phytase Citrobacter braakii gene

expressed in Asperigillus oryzae Ronozyme NP DSM

Novozymes 6-phytase Peniphora lycii gene expressed in Asperigillusoryzae Ronozyme P DSM

Novozymes 6-phytase Peniphora lycii gene expressed in Asperigillusoryzae

3-phytase Penicillium funiculosum

indicates data missing or illegible when filed

In one embodiment the phytase is a Citrobacter phytase derived from e.g.Citrobacter freundii, preferably C. freundii NCIMB 41247 and variantsthereof e.g. as disclosed in WO2006/038062 (incorporated herein byreference) and WO2006/038128 (incorporated herein by reference).Citrobacter braakii YH-15 as disclosed in WO 2004/085638, Citrobacterbraakii ATCC 51113 as disclosed in WO2006/037328 (incorporated herein byreference), as well as variants thereof e.g. as disclosed inWO2007/112739 (incorporated herein by reference) and WO2011/117396(incorporated herein by reference), Citrobacter amalonaticus, preferablyCitrobacter amalonaticus ATCC 25405 or Citrobacter amalonaticus ATCC25407 as disclosed in WO2006037327 (incorporated herein by reference),Citrobacter preferably Citrobacter gillenii DSM 13694 as disclosed inWO2006037327 (incorporated herein by reference), or Citrobacterintermedius, Citrobacter koseri, Citrobacter murliniae, Citrobacterrodentium, Citrobacter sedlakii, Citrobacter werkmanii, Citrobacteryoungae, Citrobacter species polypeptides or variants thereof.

In some embodiments, the phytase is an E. coli phytase marketed underthe name Phyzyme XP™ Danisco A/S. Alternatively, the phytase may be aButtiauxella phytase, e.g. a Buttiauxella agrestis phytase, for example,the phytase enzymes taught in WO 2006/043178, WO 2008/097619,WO2009/129489, WO2008/092901, PCT/US2009/41011 or PCT/IB2010/051804, allof which are incorporated herein by reference.

In one embodiment, the phytase may be a phytase from Hafnia, e.g. fromHafnia alvei, such as the phytase enzyme(s) taught in U.S.2008263688,which reference is incorporated herein by reference. In one embodiment,the phytase may be a phytase from Aspergillus, e.g. from Apergillusorzyae. In one embodiment, the phytase may be a phytase fromPenicillium, e.g. from Penicillium funiculosum.

Preferably, the phytase is present in the feedstuff in range of about200 FTU/kg to about 1000 FTU/kg feed, more preferably about 300 FTU/kgfeed to about 750 FTU/kg feed, more preferably about 400 FTU/kg feed toabout 500 FTU/kg feed. In one embodiment, the phytase is present in thefeedstuff at more than about 200 FTU/kg feed, suitably more than about300 FTU/kg feed, suitably more than about 400 FTU/kg feed. In oneembodiment, the phytase is present in the feedstuff at less than about1000 FTU/kg feed, suitably less than about 750 FTU/kg feed. Preferably,the phytase is present in the feed additive composition in range ofabout 40 FTU/g to about 40,000 FTU/g composition, more preferably about80 FTU/g composition to about 20,000 FTU/g composition, and even morepreferably about 100 FTU/g composition to about 10,000 FTU/gcomposition, and even more preferably about 200 FTU/g composition toabout 10,000 FTU/g composition. In one embodiment, the phytase ispresent in the feed additive composition at more than about 40 FTU/gcomposition, suitably more than about 60 FTU/g composition, suitablymore than about 100 FTU/g composition, suitably more than about 150FTU/g composition, suitably more than about 200 FTU/g composition. Inone embodiment, the phytase is present in the feed additive compositionat less than about 40,000 FTU/g composition, suitably less than about20,000 FTU/g composition, suitably less than about 15,000 FTU/gcomposition, suitably less than about 10,000 FTU/g composition.

It will be understood that as used herein 1 FTU (phytase unit) isdefined as the amount of enzyme required to release 1 μmol of inorganicorthophosphate from a substrate in one minute under the reactionconditions defined in the ISO 2009 phytase assay—A standard assay fordetermining phytase activity and 1 FTU can be found at InternationalStandard ISO/DIS 30024: 1-17, 2009. In one embodiment, the enzyme isclassified using the E.C. classification above, and the E.C.classification designates an enzyme having that activity when tested inthe assay taught herein for determining 1 FTU.

C. DFM Formulations

In one embodiment, the DFM (such as any of the multi-strain DFMcompositions disclosed herein) and, optionally, exogenous enzymes may beformulated as a liquid, a dry powder or a granule. In one embodiment,the DFMs and exogenous enzymes can be formulated as a single mixture. Inanother embodiment, the DFMs and the exogenous enzymes can be formulatedas separate mixtures. In still another embodiment, separate mixtures ofDFMs and the exogenous enzymes can be administered at the same time orat different times. In still another embodiment, separate mixtures ofDFMs and the exogenous enzymes can be administered simultaneously orsequentially. In yet another embodiment, a first mixture comprising DFMscan be administered followed by a second mixture comprising exogenousenzymes. In still another embodiment, a first mixture comprisingexogenous enzymes can be administered followed by a second mixturecomprising DFMs.

The dry powder or granules may be prepared by means known to thoseskilled in the art, such as, in top-spray fluid bed coater, in a buttomspray Wurster or by drum granulation (e.g. High sheer granulation),extrusion, pan coating or in a microingredients mixer.

In another embodiment, the DFM and/or the enzyme(s) may be coated, forexample encapsulated. Suitably the DFM and enzymes may be formulatedwithin the same coating or encapsulated within the same capsule.Alternatively, one or more of the enzymes may be formulated within thesame coating or encapsulated within the same capsule while the DFM canbe formulated in a separate coating from the enzymes.

In some embodiments, such as where the DFM is capable of producingendospores, the DFM may be provided without any coating. In suchcircumstances, the DFM endospores may be simply admixed with one or moreenzymes. In the latter case, the enzymes may be coated, e.g.encapsulated, for instance one or more or all of the enzymes may becoated, e.g. encapsulated. The enzymes may be encapsulated as mixtures(i.e. comprising one or more, two or more, three or more or all) ofenzymes or they may be encapsulated separately, e.g. as single enzymes.In one preferred embodiment, all enzymes may be coated, e.g.encapsulated, together. hi one embodiment, the coating protects theenzymes from heat and may be considered a thermoprotectant.

In another embodiment, the DFMs and exogenous feed enzymes may be mixedwith feed or administered in the drinking water, such as via awaterline. hi one embodiment, the dosage range for inclusion into wateris about 1×10³ CFU/animal/day to about 1×10¹⁵ CFU/animal/day, forexample, about 1×10³ CFU/animal/day, 1×10⁴ CFU/animal/day, 1×10⁵CFU/animal/day, 1×10⁶ CFU/animal/day, 1×10⁴ CFU/animal/day, 1×10⁸CFU/animal/day, 1×10⁹ CFU/animal/day 1×10¹⁰CFU/animal/day, 1×10¹¹CFU/animal/day, 1×10¹² CFU/animal/day, 1×10¹³ CFU/animal/day, 1×10¹⁴CFU/animal/day, or 1×10¹⁵ CFU/animal/day, inclusive of all dosagesfalling in between these values.

D. Feed Additive Compositions

In one embodiment, provided herein are feed additive compositionscomprising one or more DFMs (such as any of the multi-strain DFMsdisclosed herein) and, optionally, one or more exogenous feed enzymes.In one embodiment, the feed additive composition can be formulated inany suitable way to ensure that the formulation comprises viable DFMsand, optionally, active enzymes.

In one embodiment, the feed additive composition may be used in the formof solid or liquid preparations or alternatives thereof. Examples ofsolid preparations include powders, pastes, boluses, capsules, ovules,pills, pellets, tablets, dusts, and granules which may be wettable,spray-dried or freeze-dried. Examples of liquid preparations include,but are not limited to, aqueous, organic or aqueous-organic solutions,suspensions and emulsions.

In another embodiment, the feed additive composition can be used in asolid form. In one embodiment, the solid form is a pelleted form. Insolid form, the feed additive composition may also contain one or moreof: excipients such as microcrystalline cellulose, lactose, sodiumcitrate, calcium carbonate, dibasic calcium phosphate and glycine;disintegrants such as starch (preferably corn, potato or tapiocastarch), sodium starch glycollate, croscarmellose sodium and certaincomplex silicates; granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),sucrose, gelatin and acacia; lubricating agents such as magnesiumstearate, stearic acid, glyceryl behenate and talc may be included.

Examples of nutritionally acceptable carriers for use in preparing theforms include, for example, water, salt solutions, alcohol, silicone,waxes, petroleum jelly, vegetable oils, polyethylene glycols. propyleneglycol, liposomes, sugars, gelatin, lactose, amylose, magnesiumstearate, talc, surfactants. silicic acid, viscous paraffin. perfumeoil, fatty acid monoglycerides and diglycerides, petroethral fatty acidesters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

In one embodiment, the feed additive composition is formulated to a drypowder or granules as described in WO2007/044968 (referred to as TPTgranules) or WO 1997/016076 or WO 1992/012645 (each of which isincorporated herein by reference).

In one embodiment, the feed additive composition may be formulated to agranule feed composition comprising: an active agent comprising one ormore DFM (such as any of the multi-strain DFM compositions disclosedherein) and, optionally, one or more exogenous feed enzyme and at leastone coating. In one embodiment, the active agent of the granule retainsactivity after processing. In one embodiment, the active agent of thegranule retains an activity level after processing selected from thegroup consisting of: 50-60% activity, 60-70% activity, 70-80% activity,80-85% activity, 85-90% activity, and 90-95% activity.

In another embodiment, the granule may contain one coating. The coatingmay comprise a moisture hydrating material that constitutes at least 55%w/w of the granule. In another embodiment, the granule may contain twocoatings. The two coatings may be a moisture hydrating coating and amoisture barrier coating. In some embodiments, the moisture hydratingcoating may be from 25% to 60% w/w of the granule and the moisturebarrier coating may be from 2% to 15% w/w of the granule. The moisturehydrating coating may be selected from inorganic salts, sucrose, starch,and maltodextrin and the moisture barrier coating may be selected frompolymers, gums, whey and starch.

In yet another embodiment, the granule may be produced using a feedpelleting process and the feed pretreatment process may be conductedbetween 70° C. and 95° C. for up to several minutes, such as between 85°C. and 95° C. In another embodiment, the granule may be produced using asteam-heated pelleting process that may be conducted between 85° C. and95° C. for up to several minutes.

In one embodiment, the granule may have a moisture barrier coatingselected from polymers and gums and the moisture hydrating material maybe an inorganic salt. The moisture hydrating coating may be between 25%and 45% w/w of the granule and the moisture barrier coating may bebetween 2% and 20% w/w of the granule.

In one embodiment, the active agent retains activity after conditionsselected from one or more of: (a) a feed pelleting process; (b) asteam-heated feed pretreatment process; (c) storage; (d) storage as aningredient in an unpelleted mixture; and (e) storage as an ingredient ina feed base mix or a feed premix comprising at least one compoundselected from trace minerals, organic acids, reducing sugars, vitamins,choline chloride, and compounds which result in an acidic or a basicfeed base mix or feed premix.

In some embodiments, the DFM (e.g. DFM endospores, for example) may bediluted using a diluent, such as starch powder, lime stone or the like.In one embodiment, the DFM and the enzymes may be in a liquidformulation suitable for consumption preferably such liquid consumptioncontains one or more of the following: a buffer, salt, sorbitol and/orglycerol. In another embodiment, the feed additive composition may beformulated by applying, e.g. spraying, the enzyme(s) onto a carriersubstrate, such as ground wheat for example.

In one embodiment, the feed additive composition may be formulated as apremix. By way of example only, the premix may comprise one or more feedcomponents, such as one or more minerals and/or one or more vitamins.

In one embodiment, the DFM and exogenous feed enzymes may be formulatedwith at least one physiologically acceptable carrier selected from atleast one of maltodextrin, limestone (calcium carbonate), cyclodextrin,wheat or a wheat component, sucrose, starch, Na2SO4, Talc, PVA,sorbitol, benzoate, sorbiate, glycerol, sucrose, propylene glycol,1,3-propane diol, glucose. parabens, sodium chloride, citrate, acetate,phosphate, calcium, metabisulfite, formate and mixtures thereof.

In another embodiment, the feed additive composition can be delivered asan aqueous suspension and/or an elixir. The feed additive compositionmay be combined with various sweetening or flavoring agents, coloringmatter or dyes, with emulsifying and/or suspending agents and withdiluents such as water, propylene glycol and glycerin, and combinationsthereof. The feed additive composition may also be delivered as anaqueous suspension through a waterline.

E. Feedstuffs

In another embodiment, provided herein are feed additive compositionscontaining any of the multi-strain DFM compositions disclosed hereinthat may be used as a feed or in the preparation of a feed. The feed maybe in the form of a solution or as a solid depending on the use and/orthe mode of application and/or the mode of administration. When used asa feed or in the preparation of a feed, such as functional feed, thefeed additive composition may be used in conjunction with one or more ofthe following: a nutritionally acceptable carrier, a nutritionallyacceptable diluent, a nutritionally acceptable excipient, anutritionally acceptable adjuvant, a nutritionally active ingredient.

In one embodiment, the feed additive composition disclosed herein isadmixed with a feed component to form a feedstuff. In one embodiment,the feed may be a fodder, or a premix thereof, a compound feed, or apremix thereof. In one embodiment, the feed additive compositiondisclosed herein may be admixed with a compound feed, a compound feedcomponent or a premix of a compound feed or to a fodder, a foddercomponent, or a premix of a fodder.

In one embodiment, fodder may be obtained from one or more of the plantsselected from: alfalfa (lucerne), barley, birdsfoot trefoil, brassicas,Chau moellier, kale, rapeseed (canola), rutabaga (swede), turnip,clover, alsike clover, red clover, subterranean clover, white clover,grass, false oat grass, fescue, Bermuda grass, brome, heath grass,meadow grasses (from naturally mixed grassland swards, orchard grass,rye grass, Timothy-grass, corn (maize), millet, oats, sorghum, soybeans,trees (pollard tree shoots for tree-hay), wheat, and legumes.

Compound feeds can be complete feeds that provide all the daily requirednutrients, concentrates that provide a part of the ration (protein,energy) or supplements that only provide additional micronutrients, suchas minerals and vitamins. The main ingredients used in compound feed arethe feed grains, which include coin, soybeans, sorghum, oats, andbarley.

A “premix,” as referred to herein, may be a composition composed ofmicro-ingredients such as vitamins, minerals, chemical preservatives,antibiotics, fermentation products, and other essential ingredients.Premixes are usually compositions suitable for blending into commercialrations.

In one embodiment, a feedstuff as disclosed herein may comprise one ormore feed materials selected from the group comprising cereals, such assmall grains (e.g., wheat, barley, rye, oats and combinations thereof)and/or large grains such as maize or sorghum; by products from cereals,such as com gluten meal, Distillers Dried Grain Solubles (DDGS), wheatbran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls,palm kernel, and citrus pulp; protein obtained from sources such assoya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fishmeal, dried plasma protein, meat and bone meal, potato protein, whey,copra, sesame: oils and fats obtained from vegetable and animal sources:and minerals and vitamins.

In yet another embodiment, a feedstuff may comprise at least one highfiber feed material and/or at least one by-product of the at least onehigh fiber feed material to provide a high fiber feedstuff. Examples ofhigh fiber feed materials include: wheat, barley, rye, oats, by productsfrom cereals, such as coin gluten meal, Distillers Dried Grain Solubles(DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, ricehulls, oat hulls, palm kernel, and citrus pulp. Some protein sources mayalso be regarded as high fiber: protein obtained from sources such assunflower, lupin, fava beans and cotton

In still another embodiment, the feed may be one or more of thefollowing: a compound feed and premix, including pellets, nuts or(cattle) cake; a crop or crop residue: coin, soybeans, sorghum, oats,barley, corn stover, copra, straw, chaff, sugar beet waste; fish meal;freshly cut grass and other forage plants; meat and bone meal; molasses;oil cake and press cake; oligosaccharides; conserved forage plants: hayand silage; seaweed; seeds and grains, either whole or prepared bycrushing, milling etc.; sprouted grains and legumes; yeast extract.

In one embodiment, the feed additive composition of disclosed herein isadmixed with the product (e.g. feedstuff). Alternatively, the feedadditive composition may be included in the emulsion or raw ingredientsof a feedstuff. In another embodiment, the feed additive composition ismade available on or to the surface of a product to be affected/treated.In still another embodiment, the feed additive compositions disclosedherein may be applied, interspersed, coated and/or impregnated to aproduct (e.g. feedstuff or raw ingredients of a feedstuff) with acontrolled amount of DFM and, optionally, enzymes.

In yet another embodiment, the DFM and optional enzymes may be usedsimultaneously (e.g. when they are in admixture together or even whenthey are delivered by different routes) or sequentially (e.g. they maybe delivered by different routes).

In one embodiment, the DFM and optional enzymes are applied to thefeedstuff simultaneously. In yet another embodiment, the DFM andoptional enzymes are admixed prior to being delivered to a feedstuff orto a raw ingredient of a feedstuff.

In one embodiment, the DFMs in the feed additive compositions disclosedherein can be added in suitable concentrations including but not limitedto concentrations in the final feed product that offer a daily dose offrom about 2×10³ CFU to about 2×10¹¹ CFU, from about 2×10⁶ to about1×10¹⁰, and from about 3.75×10⁷ CFU to about 1×10¹⁰CFU.

III. Methods A. Methods for Improving Performance Metrics in an Animal

Further provided herein are methods for increasing performance metricsof an animal. In another embodiment, the disclosure relates to methodsof increasing performance metrics of a bird. In still anotherembodiment, the disclosure relates to methods of increasing performancemetrics of poultry, including but not limited to broilers, chickens andturkeys.

In yet another embodiment, the disclosure relates to a method comprisingadministering to an animal a composition comprising DFMs (such as any ofthe multi-strain DFMs disclosed herein) and, optionally, exogenous feedenzymes. In still another embodiment, the disclosure relates to a methodcomprising administering to an animal an effective amount of acomposition comprising DFMs and optional exogenous feed enzymes toincrease performance of the animal. This effective amount can beadministered to the animal in one or more doses. In one embodiment, theanimal is poultry. In still another embodiment, the animal is a broiler.

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optionally exogenousfeed enzymes to increase average daily feed intake. In some embodiments,the average daily feed intake increases by any of about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, 105%, or 110%, inclusive of all values falling in betweenthese percentages, relative to animals who are not administered one ormore of the multi-strain DFM compositions disclosed herein. In someembodiments, the composition is a feed additive composition. In otherembodiments, the composition is a feed or feedstuff.

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optional exogenousfeed enzymes to increase average daily weight gain. In some embodiments,the average daily weight gain increases by any of about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, 105%, or 110%, inclusive of all values falling in betweenthese percentages, relative to animals who are not administered one ormore of the multi-strain DFM compositions disclosed herein. In someembodiments, the composition is a feed additive composition. In otherembodiments, the composition is a feed or feedstuff.

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optional exogenousfeed enzymes to increase total weight gain. In some embodiments, totalweight gain increases by any of about 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or110%, inclusive of all values falling in between these percentages,relative to animals who are not administered one or more of themulti-strain DFM compositions disclosed herein. In some embodiments, thecomposition is a feed additive composition. In other embodiments, thecomposition is a feed or feedstuff.

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optional exogenousfeed enzymes to increase feed conversion, which can be measured byeither feed:gain or gain:feed. In some embodiments, feed conversionincreases by any of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%,inclusive of all values falling in between these percentages, relativeto animals who are not administered one or more of the multi-strain DFMcompositions disclosed herein. In some embodiments, the composition is afeed additive composition. In other embodiments, the composition is afeed or feedstuff.

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optional exogenousfeed enzymes to increase feed efficiency. In some embodiments, feedefficiency increases by any of about 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or110%, inclusive of all values falling in between these percentages,relative to animals who are not administered one or more of themulti-strain DFM compositions disclosed herein. In some embodiments, thecomposition is a feed additive composition. In other embodiments, thecomposition is a feed or feedstuff

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optional exogenousfeed enzymes to decrease mortality. In some embodiments, mortalitydecreases by any of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, inclusive of allvalues falling in between these percentages, relative to animals who arenot administered one or more of the multi-strain DFM compositionsdisclosed herein. In some embodiments, the composition is a feedadditive composition. In other embodiments, the composition is a feed orfeedstuff.

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optional exogenousfeed enzymes to decrease feed conversion ratio (FCR). In someembodiments, FCR decreases by any of about 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. or 100%,inclusive of all values falling in between these percentages, relativeto animals who are not administered one or more of the multi-strain DFMcompositions disclosed herein. In some embodiments, the composition is afeed additive composition. In other embodiments, the composition is afeed or feedstuff

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optional exogenousfeed enzymes to increase gut barrier integrity. In some embodiments, gutbarrier integrity increases by any of about 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,105%, or 110%, inclusive of all values falling in between thesepercentages, relative to animals who are not administered one or more ofthe multi-strain DFM compositions disclosed herein. “Gut barrierintegrity” can refer to, without limitation, epithelial damage andepithelial penneability which is characterized by a shortening of villi,a lengthening of crypts and an infiltration of inflammatory cells (suchas, without limitation, CD3+ cells). The latter damage and inflammationmarkers can also be associated with a “severe” macroscopic appearance ofthe gut—compared to a “normal” appearance—when evaluated using a scoringsystem such as the one described by Teirlynck et al. (2011). In someembodiments, the composition is a feed additive composition. In otherembodiments, the composition is a feed or feedstuff.

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optional exogenousfeed enzymes to decrease or prevent pathogen infection (such as, withoutlimitation, infection by Clostridium perfringens, Campylobacter jejuni,a Salmonella sp., and/or Escherichia coli). In some embodiments,pathogen infection decreases by any of about 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%, inclusive of all values falling in between these percentages,relative to animals who are not administered one or more of themulti-strain DFM compositions disclosed herein. In some embodiments, thecomposition is a feed additive composition. In other embodiments, thecomposition is a feed or feedstuff. In some embodiments, the compositionis a feed additive composition. In other embodiments, the composition isa feed or feedstuff.

In another embodiment, the disclosure relates to a method comprisingadministering to an animal (such as a domesticated bird, for example, achicken) an effective amount of a composition comprising DFMs (such asany of the multi-strain DFMs disclosed herein) and optional exogenousfeed enzymes to decrease or prevent pathogen shedding in the feces (suchas, without limitation, shedding of Clostridium perfringens,Campylobacter jejuni, a Salmonella sp., and/or Escherichia coli). Insome embodiments, pathogen shedding in the feces decreases by any ofabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100%, inclusive of all values falling inbetween these percentages, relative to animals who are not administeredone or more of the multi-strain DFM compositions disclosed herein. Insome embodiments, the composition is a feed additive composition. Inother embodiments, the composition is a feed or feedstuff.

In still another embodiment, the DFM composition (such as a feed or feedadditive composition) administered to the animal (such as a domesticatedbird, for example, a chicken) is a multi-strain DFM comprising one ormore of L. reuteri strain S 1 (CBS 145921), or a strain having all ofthe identifying characteristics of L. reuteri strain S1, or a microbehaving a 16S ribosomal RNA sequence displaying at least about 97.0%sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%,99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequence ofL. reuteri strain S1 (SEQ ID NO:7); L. reuteri strain S2 (CBS 145922),or a strain having all of the identifying characteristics of L. reuteristrain S2, or a microbe having a 16S ribosomal RNA sequence displayingat least about 97.0% sequence similarity (such as any of about 97%,97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to a 16Sribosomal RNA sequence of L. reuteri strain S2 (SEQ ID NO:8): and/or L.reuteri strain S3 (CBS 145923), or a strain having all of theidentifying characteristics of L. reuteri strain S3, or a microbe havinga 16S ribosomal RNA sequence displaying at least about 97.0% sequencesimilarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or100% sequence similarity) to a 16S ribosomal RNA sequence of L. reuteristrain S3 (SEQ ID NO:9). In another embodiment, the DFM compositionincludes L. reuteri strains S1 and S2; L. reuteri strains S1 and S3; L.reuteri strains S2 and S3; or L. reuteri strains S1. S2, and S3. In someembodiments, the one or more (such as 1, 2, or 3) L. reuteri strain(s)is (are) administered to an animal at a rate of at least 1×10⁴CFU/animal/day. For poultry, according to one non-limiting embodiment,the one or more L. reuteri strain(s) can be fed at about 1×10⁵ CFU/gfeed to about 1×10¹⁰ CFU/g feed. In at least some embodiments, the oneor more L. reuteri strains is (are) fed at about 1×10⁵ CFU/bird/day orabout 1 ×10⁸ CFU/bird/day.

In still another embodiment, the DFM composition (such as a feed or feedadditive composition) administered to the animal (such as a domesticatedbird, for example, a chicken) is a multi-strain DFM comprising one ormore of L. gallinarum strain H1 (CBS 145918), or a strain having all ofthe identifying characteristics L. gallinarum strain H1, or a microbehaving a 16S ribosomal RNA sequence displaying at least about 97.0%sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%,99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequence ofL. gallinarum strain H1 (SEQ ID NO:11); L. salivarius strain H2 (CBS145919), or a strain having all of the identifying characteristics of L.salivarius strain H2, or a microbe having a 16S ribosomal RNA sequencedisplaying at least about 97.0% sequence similarity (such as any ofabout 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity)to a 16S ribosomal RNA sequence of L. salivarius strain H2 (SEQ IDNO:10); L. agilis strain H3, or a strain having all of the identifyingcharacteristics of L. agilis strain 113, or a microbe having a 16Sribosomal RNA sequence displaying at least about 97.0% sequencesimilarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or100% sequence similarity) to a 16S ribosomal RNA sequence of L. agilisstrain H3 (SEQ ID NO;1); and/or L. reuteri strain A2 (CBS 145924), or astrain having all of the identifying characteristics of L. reuteristrain A2, or a microbe having a 16S ribosomal RNA sequence displayingat least about 97.0% sequence similarity (such as any of about 97%,97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to a 16Sribosomal RNA sequence of L. reuteri strain A2 (SEQ ID NO:12). In someembodiments, the DFM composition includes only L. gallinarum strain H1,L. salivarius strain H2, L. agilis strain H3, or L. reuteri strain A2.In another embodiment, the DFM composition includes L. gallinarum strainH1 and L. salivarius strain H2; L. reuteri strain A2 and L. agilisstrain H3; L. gallinarum strain H1 and L. reuteri strain A2; L.salivarius strain H2 and L. agilis strain H3; L. salivarius strain H2and L. reuteri strain A2; L. agilis strain H3 and L. reuteri strain A2;L. gallinarum strain H1, L. salivarius strain H2, and L. agilis strainH3; L. gallinarum strain H1, L. agilis strain H3, and L. reuteri strainA2; L. salivarius strain H2, L. agilis strain H3, and L. reuteri strainA2; L. gallinarum strain H1, L. salivarius strain H2, L. agilis strainH3, and L. reuteri strain A2; L. gallinarum strain H1, L. salivariusstrain H2, and L. agilis strain H3; or L. gallinarum strain H1, L.salivarius strain H2, and L. reuteri strain A2. In some embodiments, L.reuteri strain A2 produces reuterin (3-hydroxypropionaldehyde). In otherembodiments, L. reuteri strain A2 does not produce reuterin(3-hydroxypropionaldehyde). In some embodiments, the one or more H1, H2,H3, and/or A2 strain(s) (such as H1, H2, and H3; or H1,H2, and A2) is(are) administered to an animal at a rate of at least 1×10⁴CFU/animal/day. For poultry, according to one non-limiting embodiment,the one or more H1, H2, H3, and/or A2 strain(s) (such as H1, H2, and H3;or H1, H2, and A2) can be fed at about 1×10⁵ CFU/g feed to about 1×10¹⁰CFU/g feed. In at least some embodiments, the one or more H1, H2, H3,and/or A2 strain(s) (such as H1, H2, and H3; or H1, H2, and A2) strainsis (are) fed at about 1×10⁵ CFU/bird/day or about 1×10⁸ CFU/bird/day.

In still another embodiment, the DFM composition (such as a feed or feedadditive composition) administered to the animal (such as a domesticatedbird, for example, a chicken) is a multi-strain DFM comprising one ormore of L. salivarius strain A1, or a strain having all of theidentifying characteristics L. salivarius strain A1, or a microbe havinga 16S ribosomal RNA sequence displaying at least about 97.0% sequencesimilarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or100% sequence similarity) to a 16S ribosomal RNA sequence of L.salivarius strain Al (SEQ ID NO:2); L. reuteri strain A2 (CBS 145924),or a strain having all of the identifying characteristics of L. reuteristrain A2, or a microbe having a 16S ribosomal RNA sequence displayingat least about 97.0% sequence similarity (such as any of about 97%,97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to a 16Sribosomal RNA sequence of L. reuteri strain A2 (SEQ ID NO:12); and/or L.agilis strain A3, or a strain having all of the identifyingcharacteristics of L. agilis strain A3, or a microbe having a 16Sribosomal RNA sequence displaying at least about 97.0% sequencesimilarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or100% sequence similarity) to a 16S ribosomal RNA sequence of L. agilisstrain A3 (SEQ ID NO:3). In some embodiments, L. reuteri strain A2produces reuterin (3-hydroxypropionaldehyde). In other embodiments, L.reuteri strain A2 does not produce reuterin (3-hydroxypropionaldehyde).In some embodiments, the DFM composition includes only L. salivariusstrain A1, L. reuteri strain A2, or L. agilis strain A3. In anotherembodiment, the DFM composition includes L. salivarius strain Aand L.reuteri strain A2; L. salivarius strain A1 and L. agilis strain A3; L.reuteri strain A2 and L. agilis strain A3, or L. salivarius strain A1,L. reuteri strain A2, and L. agilis strain A3. In some embodiments, theone or more A1, A2 and/or A3 strain(s) is (are) administered to ananimal at a rate of at least 1×10⁴ CFU/animal/day. For poultry,according to one non-limiting embodiment, the one or more A1, A2 and/orA3 strain(s) can be fed at about 1×10⁵ CFU/g feed to about 1×10¹⁰ CFU/gfeed. In at least some embodiments, the one or more A1, A2 and/or A3strains is (are) fed at about 1×10⁵ CFU/bird/day or about 1×10⁸CFU/bird/day.

In still another embodiment, the DFM composition (such as a feed or feedadditive composition) administered to the animal (such as a domesticatedbird, for example, a chicken) is a multi-strain DFM comprising one ormore of L. agilis strain D1, or a strain having all of the identifyingcharacteristics L. agilis strain D1, or a microbe having a 16S ribosomalRNA sequence displaying at least about 97.0% sequence similarity (suchas any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequencesimilarity) to a 16S ribosomal RNA sequence of L. agilis strain D1 (SEQID NO:5), L. salivarius strain D2, or a strain having all of theidentifying characteristics of L. salivarius strain D2, or a microbehaving a 16S ribosomal RNA sequence displaying at least about 97.0%sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%,99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequence ofL. salivarius strain D2 (SEQ ID NO:4); and/or L. crispatus strain D3, ora strain having all of the identifying characteristics of L. crispatusstrain D3, or a microbe having a 16S ribosomal RNA sequence displayingat least about 97.0% sequence similarity (such as any of about 97%,97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to a 16Sribosomal RNA sequence of L. crispatus strain D3 (SEQ ID NO:6). In someembodiments, the DFM composition includes only L. agilis strain Dl, L.salivarius strain D2, or L. crispatus strain D3. In another embodiment,the DFM composition includes L. agilis strain D1 and L. salivariusstrain D2; L. agilis strain D1 and L. crispatus strain D3; L. salivariusstrain D2 and L. crispatus strain D3; or L. agilis strain D1, L.salivarius strain D2, and L. agilis strain A3. In some embodiments, theone or more D1, D2 and/or D3 strain(s) is (are) administered to ananimal at a rate of at least 1×10⁴ CFU/animal/day. For poultry,according to one non-limiting embodiment, the one or more D1, D2 and/orD3 strain(s) can be fed at about 1×10⁵ CFU/g feed to about 1×10¹⁰ CFU/gfeed. In at least some embodiments, the one or more D1, D2 and/or D3strains is (are) fed at about 1×10⁵ CFU/bird/day or about 1×10⁸CFU/bird/day.

The DFM compositions provided herein can be administered, for example,as a strain-containing culture solution, a strain-containingsupernatant, or a bacterial product of a culture solution.Administration of a composition comprising a DFM and optional exogenousfeed enzymes provided herein to an animal can increase the performanceof the animal. In one embodiment, administration of a DFM providedherein to an animal can increase the average daily feed intake (ADFI),average daily gain (ADG), or feed efficiency (gain:feed; G:F)(collectively, “performance metrics”). One or more than one of theseperformance metrics may be improved.

The composition comprising DFMs and exogenous feed enzymes may beadministered to the animal in one of many ways. For example, thecomposition can be administered in a solid form as a veterinarypharmaceutical, may be distributed in an excipient, preferably water(such as through a waterline), and directly fed to the animal, may bephysically mixed with feed material in a dry form, or the compositionmay be formed into a solution and thereafter sprayed onto feed material.The method of administration of the compositions disclosed herein to theanimal is considered to be within the skill of the artisan.

When used in combination with a feed material, the feed material caninclude corn, soybean meal, byproducts like distillers dried grains withsolubles (DDGS), and vitamin/mineral supplement. Other feed materialscan also be used.

Thus, in at least some embodiments, the effective amount of thecomposition comprising DFMs and optional exogenous feed enzymes isadministered to an animal by supplementing a feed intended for theanimal. As used herein, “supplementing,” refers to the action ofincorporating the effective amount of bacteria provided herein directlyinto the feed intended for the animal. Thus, the animal, when feeding,ingests the bacteria provided herein.

B. Methods for Preparing a Feed Additive Composition

Also provided herein are methods for preparing a feed additivecomposition comprising combining two or more of the DFMs disclosedherein. hi some embodiments, the method includes combining two or more(such as any of 2 or 3) of L. reuteri strain S1 (CBS 145921), or astrain having all of the identifying characteristics of L. reuteristrain S1, or a microbe having a 16S ribosomal RNA sequence displayingat least about 97.0% sequence similarity (such as any of about 97%,97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to a 16Sribosomal RNA sequence of L. reuteri strain S1 (SEQ ID NO:7); L. reuteristrain S2 (CBS 145922), or a strain having all of the identifyingcharacteristics of L. reuteri strain S2, or a microbe having a 16Sribosomal RNA sequence displaying at least about 97.0% sequencesimilarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or100% sequence similarity) to a 16S ribosomal RNA sequence of L. reuteristrain S2 (SEQ ID NO:8); L. reuteri strain S3 (CBS 145923), or a strainhaving all of the identifying characteristics of L. reuteri strain S3,or a microbe having a 16S ribosomal RNA sequence displaying at leastabout 97.0% sequence similarity (such as any of about 97%, 97.5%, 98%,98.5%, 99%, 99.5%, or 100% sequence similarity) to a 16S ribosomal RNAsequence of L. reuteri strain S3 (SEQ ID NO:9). In another embodiment,L. reuteri strains S1 and S2 are combined; L. reuteri strains S1 and S3are combined; L. reuteri strains S2 and S3 are combined; or A.colihominis strains S1, S2, and S3 are combined.

In yet further embodiments, the method includes combining two or more(such as any of 2, 3, or 4) of L. gallinarum strain H1 (CBS 145918), ora strain having all of the identifying characteristics L. gallinarumstrain H1, or a microbe having a 16S ribosomal RNA sequence displayingat least about 97.0% sequence similarity (such as any of about 97%,97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to a 16Sribosomal RNA sequence of L. gallinarum strain H1 (SEQ ID NO:11); L.salivarius strain H2 (CBS 145919), or a strain having all of theidentifying characteristics of L. salivarius strain H2, or a microbehaving a 16S ribosomal RNA sequence displaying at least about 97.0%sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%,99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequence ofL. salivarius strain H2 (SEQ ID NO:10); L. agilis strain H3, or a strainhaving all of the identifying characteristics of L. agilis strain H3, ora microbe having a 16S ribosomal RNA sequence displaying at least about97.0% sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%,99%, 99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequenceof L. agilis strain H3 (SEQ ID NO:1); and/or L. reuteri strain A2 (CBS145924), or a strain having all of the identifying characteristics of L.reuteri strain A2, or a microbe having a 16S ribosomal RNA sequencedisplaying at least about 97.0% sequence similarity (such as any ofabout 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity)to a 16S ribosomal RNA sequence of L. reuteri strain A2 (SEQ ID NO:12).In another embodiment, L. gallinarum strain H1 and L. salivarius strainH2 are combined; L. gallinarum strain H1 and L. agilis strain H3 arecombined; L. gallinarum strain H1 and L. reuteri strain A2 are combined;L. salivarius strain H2 and L. agilis strain H3 are combined; L.salivarius strain H2 and L. reuteri strain A2 are combined; L. agilisstrain H3 and L. reuteri strain A2 are combined; L. gallinarum strainH1, L. salivarius strain H2, and L. agilis strain H3 are combined; L.gallinarum strain H1, L. agilis strain H3. and L. reuteri strain A2 arecombined; L. salivarius strain H2. L. agilis strain H3, and L. reuteristrain A2 are combined; L. gallinarum strain H1, L. salivarius strain H2L. agilis strain H3, and L. reuteri strain A2 are combined; L.gallinarum strain H1, L. salivarius strain H2, and L. agilis strain H3are combined; or L. gallinarum strain H1, L. salivarius strain H2 and L.reuteri strain A2 are combined.

In additional embodiments, the method includes combining two or more(such as any of 2 or 3) of L. salivarius strain A1, or a strain havingall of the identifying characteristics L. salivarius strain A1, or amicrobe having a 16S ribosomal RNA sequence displaying at least about97.0% sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%,99%, 99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequenceof L. salivarius strain A1 (SEQ ID NO:2); L. reuteri strain A2 (CBS145924), or a strain having all of the identifying characteristics of L.reuteri strain A2, or a microbe having a 16S ribosomal RNA sequencedisplaying at least about 97.0% sequence similarity (such as any ofabout 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity)to a 16S ribosomal RNA sequence of L. reuteri strain A2 (SEQ ID NO:12);and/or L. agilis strain A3, or a strain having all of the identifyingcharacteristics of L. agilis strain A3, or a microbe having a 16Sribosomal RNA sequence displaying at least about 97.0% sequencesimilarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or100% sequence similarity) to a 16S ribosomal RNA sequence of L. agilisstrain A3 (SEQ ID NO:3). In another embodiment, L. salivarius strain A1and L. reuteri strain A2 are combined; L. salivarius strain A1 and L.agilis strain A3 are combined; L. reuteri strain A2 and L. agilis strainA3 are combined; or L. salivarius strain A1, L. reuteri strain A2, andL. agilis strain A3 are combined.

In further embodiments, the method includes combining two or more (suchas any of 2 or 3) of L. agilis strain D1, or a strain having all of theidentifying characteristics L. agilis strain D1, or a microbe having a16S ribosomal RNA sequence displaying at least about 97.0% sequencesimilarity (such as any of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or100% sequence similarity) to a 16S ribosomal RNA sequence of L. agilisstrain D1 (SEQ ID NO:5); L. salivarius strain D2, or a strain having allof the identifying characteristics of L. salivarius strain D2, or amicrobe having a 16S ribosomal RNA sequence displaying at least about97.0% sequence similarity (such as any of about 97%, 97.5%, 98%, 98.5%,99%, 99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequenceof L. salivarius strain D2 (SEQ ID NO:4); and/or L. crispatus strain D3,or a strain having all of the identifying characteristics of L.crispatus strain D3, or a microbe having a 16S ribosomal RNA sequencedisplaying at least about 97.0% sequence similarity (such as any ofabout 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity)to a 16S ribosomal RNA sequence of L. crispatus strain D3 (SEQ ID NO:6).In another embodiment, L. agilis strain D1 and L. salivarius strain D2are combined; L. agilis strain D1 and L. crispatus strain D3 arecombined; L. salivarius strain D2 and L. crispatus strain D3 arecombined; or L. agilis strain D1, L. salivarius strain D2, and L.crispatus strain D3 are combined.

Additionally, the methods for preparing a feed additive composition canfurther include combining the feed additive composition with one or moreof the exogenous enzymes disclosed herein (for example, one or more of aphytase, a protease, an amylase, a xylanase or a beta-glucanase). Themethod can additionally include a further step of packaging the feedadditive composition for storage or transport.

C. Methods for Removing Antimicrobial Resistance (AMR) Genes

Bacteria have evolved to overcome a wide range of antibiotics, andresistance mechanisms against most of the conventional antibiotics havebeen identified in some bacteria. Accelerated development of newerantibiotics is being overrun by the pace of bacterial resistance. In theUSA, for example, over 70% of hospital-acquired infections involvebacteria resistant to at least one antibiotic, and in Japan over 50% ofthe clinical isolates of Staphylococcus aureus are multidrug-resistant.Thus, removal of antimicrobial resistance (AMR) genes fromprobiotics/DFMs used for the prevention of intestinal inflammation andin the maintenance of intestinal homeostasis can in some instances bedesirable.

AMR genes carried by a variety of bacteria are known in the art and thesequences of antibiotic resistance genes in any particular bacteria canbe determined if desired. In certain non-limiting embodiments, thepresent disclosure includes CRISPR systems which comprise spacersencoding targeting RNA that is directed to bacterial DNA sequences whichcomprise antibiotic resistance genes. In some embodiments, theresistance gene confers resistance to a narrow-spectrum beta-lactamantibiotic of the penicillin class of antibiotics. In other embodiments,the resistance gene confers resistance to methicillin (e.g., methicillinor oxacillin), or flucloxacillin, or dicloxacillin, or some or all ofthese antibiotics.

Examples of AMR genes include, but are not limited to, fosfomycinresistance gene fosB, tetracycline resistance gene tetM, kanamycinnucleotidyltransferase aadD, bifunctional aminoglycoside modifyingenzyme genes aacA-aphD, chloramphenicol acetyltransferase cat,mupirocin-resistance gene ileS2, vancomycin resistance genes vanX, vanR,vanH, vraE, vraD, methicillin resistance factor femA, fmtA, mecl,streptomycin adenylyltransferase spc1, spc2, anti, ant2, pectinomycinadenyltransferase spd, ant9, aadA2, InuC, vatE, tetW, and any otherresistance gene. In some embodiments, the AMRs are associated with oneor more mobile genetic elements (such as a transposon) than can belocated near the AMR gene (such as within any of about 10 kb, 9 kb, 8kb, 7 kb, 6 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or closer to theAMR gene, including distances falling in between any of these values).

AMRs can be identified or assayed qualitatively and/or quantitatively byany number of methods known in the art. For example, DNA can beextracted from microbial samples and assessed for the presence ofantibiotic resistance genes using, e.g., quantitative PCR (qPCR),including multiplex quantitative PCR (qPCR). The microbial DNA arrayassay may test for a plurality of antibiotic resistance genes andfacilitate testing for highly prevalent bacterial antibiotic resistancegenes in a single reaction with both resistance gene identification(present vs absent) and quantitative profiling (expression relative toan internal standard). Other methods for identifying AMRs include,without limitation, whole genome sequencing via use of short-readsequencing technology (Illumina) and/or long-read sequencing technologyplatforms such as Oxford Nanopore Technology or PacBio or via thetechniques used and discussed in Example 5. Once identified, AMR genescan be removed or inactivated using any number of standard moleculartools available in the art such as recombineering (Zhang et al., 2018, JBacteriol 200; Ozcam et al.. 2019, Appl Environ Microbiol 85. thedisclosures of which are incorporated by reference herein), or CRISPRbased methods (Oh & Van Pijkeren, 2014. Nucleic Acids Res 42:e131; U.S.Patent Application Publication No. 2017/0260546, the disclosures ofwhich are incorporated by reference herein).

In some embodiments, one or more of (such as any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or 11) the strains provided herein including L. reuteri strainS1 (CBS 145921), L. reuteri strain S2 (CBS 145922), L. reuteri strain S3(CBS 145923), L. gallinarum strain H1 (CBS145918), L. salivarius strainH2 (CBS 145919), L. reuteri strain A2 (CBS 145924), L. agilis strain H3,L. salivarius strain A1, L. agilis strain A3, L. agilis strain D1, L.salivarius strain D2, and L. crispatus strain D3 further comprise one ormore (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) inactivatedor deleted AMR genes.

IV. Kits

Further provided herein are kits containing one or more of the DFMs(such as one or more of the multi-strain DFMs) disclosed herein. Thekits can include one or more of (such as any of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or 11) the strains provided herein including L. reuteri strain S1(CBS 145921), L. reuteri strain S2 (CBS 145922), L. reuteri strain S3(CBS 145923), L. gallinarum strain H1 (CBS145918), L. salivarius strainH2 (CBS 145919), L. reuteri strain A2 (CBS 145924), L. agilis strain H3,L. salivarius strain A1, L. agilis strain A3, L. agilis strain D1, L.salivarius strain D2, and L. crispatus strain D3 along with instructionsfor proper storage, maintenance, and use for administering to an animalto improve one or more performance metrics. In one embodiment, the kitcan include strains S1, S2, and/or S3. In another embodiment, the kitcan include strains H1, H2, H3, and/or A2 (such as H1, H2, and H3; orH1, H2, and A2). In a further embodiment, the kit can include strainsA1, A2, and/or A3. In yet a further embodiment, the kit can includestrains D1, D2, and/or D3. The kits can additionally include one or moreof the exogenous enzymes disclosed herein (for example, one or more of aphytase, a protease, an amylase, a xylanase or a beta-glucanase).

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

EXAMPLES Example 1: Materials and Methods

In the following examples, various methods and assays were used as setforth below for ease in reading. Any deviations from the protocolsprovided below are indicated in the relevant sections.

Isolation of Lactobacillus Strains from Chicken Intestinal Tracts

GIT Samples dissected in the field were transferred into anaerobictransport media as quickly as possible. Strain isolation started onceintestinal samples arrived at the lab. Ceca, ileum, jejunum or duodemunsamples were dissected using sterile technique inside an anaerobicchamber. The digesta was discarded and mucosal-bound material wasscraped using a loop. This material was then transferred into sterilemedia or buffer and serially diluted. Serial dilutions ranging from 10⁻¹to 10⁻⁶ were plated onto petri dishes or omni plates of various mediatypes using plating beads. These plates were then incubatedanaerobically until colonies became visible. De Man, Rogosa and Sharpeagar (MRS) or Brain heart infusion medium (BHI) was used for isolation.

Once colonies were visible on an agar plate, colonies were picked in theanaerobic chamber to liquid media in a 96 deep well plate. Some plateswere initially picked into a small volume of liquid media (i.e. 200 μl)and then media added to 800 μl 1-4 days later to increase growth. Colonypicking could be done at multiple time points for the same plate. Forexample, large colonies were picked on day 2, and then very smallcolonies or new colonies were picked at day 5.

Analysis of the Microbial Composition of Mucosa Region of the SmallIntestines by 16S Sequencing and netB qPCR

To evaluate the microbial composition of the small intestines, chickenswabs of the mucosa region of the small intestines was analyzed by 16Ssequencing. Chicken gastro intestinal tract (GIT) is removed andseparated into four sections: duodenum, jejunum, ileum and ceca. Eachsection is squeezed to remove the digesta contents and then cutlongitudinally to expose the inner surface. Each section inner surfaceis swabbed with a FLOQSwab (Copan Mfgr, Murrieta, Calif.). The swab isadded directly to a well of a 96 well Qiagen MagAttract PowerSoil kit(Qiagen, Hilden, Germany). The swabs were processed for bacterial DNAisolation as per the manufacturer's instructions using the KingFisherFlex automation platform. Isolated metagenomic DNA is then ready for NGSsample preparation

Metagenomic DNA purified from chicken GIT swabs was prepared for 16Scommunity sequencing as follows: DNA is diluted 1:5 by adding 20 μl ofmolecular biology grade water to 5 μl of purified DNA at 0.1- 10 ng/μl.Then 2 μl of the diluted DNA was added to a PCR reaction along with 25μl of ABI Universal TaqMan Reaction mix without UNG (ThermoFisher#4326614), 0.1 μl each PCR primers at 100 uM and 24.8 μl of MolecularBiology Grade water for a total volume of 50 μl. The PCR primers werethe Illtunina-V4-515F-RJ:TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA andIllumina-V4-806R-RJ:GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT were used. ThePCR reactions were: 10 min at 95° C. followed by 35 cycles of 95° C. 15sec+55° C. 30 sec+72° C. for 2 min. Amplified reactions were purifiedusing Ampure XP Magnetic Beads (Beckman Coulter A63881) as permanufacturer's instructions using the Agilent Bravo Automated RoboticWorkstation. 2 μl of each amplicon pool was then indexed in a second PCRreaction using the same conditions as above with Illumina XT IndexPrimers (Illumina XT v2.0 #FC-131-2001-2004) for 15 cycles. Indexedamplicons are then pooled and purified with AmPure XP Magnetic Beads onthe Agilent Bravo Automated Robotic Workstation. Pooled, indexedamplicons were quantitated using the Kapa Illumina LibraryQuantification Kit (KAPA #KK4835) as per manufacturer's instructions.Purified, quantitated, indexed, pools were loaded on the Illumina MiSeqat a final concentration of 8 pM along with 15% Illumina PhiX (IlluminaFC-110-3001). Sequencing was run for 2×250 Paired End cycles.

The 16S Amplicon data from Illumina Miseq sequencing were analyzed.Paired-end reads were first merged by Flash (Mogoc et al.,Bioinformatics. 2011;27:2957-63). The forward and reverse primers wereremoved from the merged reads, and reads with overall quality score lessthan 20 were discarded by RDP Initial Process tool (Fish et al., FrontMicrobiol. 2013;4:291). This step also removes reads originated fromchicken mitochondria due to their length shorter than 200 bp. In thenext step reads were assigned to bacterial and archaeal taxonomy by RDPClassifier (Wang et al., Appl Environ Microbiol. 73(16):5261-5267). Thereads passed the above quality processing steps were clustered at 98% byCD-HIT (Li & Godzik, Bioinformatics, 2006: 22:1658-9) to obtainOperational Taxonomic Units (OTU). The representative sequence from eachOTU was assigned to the closest species by RDP pairwise alignment tool(Fish et al., Front Microbiol. 2013; 4:291) against a vetted 16Sreference database containing mostly 16S genes from type strains andpublic genomes. The relative abundance of an OTU in a sample was thefraction of reads assigned to that OTU. An OTU was assigned to a speciesif it has at least 98% identity to that species in the referencedatabase. The average abundance of a species in a consortium in smallintestine was calculated as the average relative abundance of thatspecies from DUO, JEJ and ILL samples of each treatment.

The same DNA preparations were also quantified for netB gene using qPCR.NetB is an important virulence factor for C. perfringens. Each assay wasrun using 1.5 μl of DNA along with 0.05 μl of TaqMan Universal MasterMix, 0.2 μl of 100 uM Forward and Reverse Primers, 0.05 μl of TaqManProbe, and 9.55 μl Molecular Biology Grade water. The qPCR reactionconditions were as follows: 10 min at 95° C.+40 cycles of 95° C. 15sec+60° C. 60 sec on the ABI Quant Studio qPCR instrument. Sample datawas quantified using genomic DNA from a netB positive C. perfringens.Primer and probe sequences used are in Table 5 below.

TABLE 5 qPCR Primers and Probes Primer Direc- Sequence Target Name tion(5′ to 3′) Total 16S-T1- for CGGTGAATA Bacteria 1369F CGTTCYCGG 16S16S-T1- rev GGWTACCTT 1492R GTTACGACT T 16S-T1- probe CTTGTACAC 1389TACCGCCCGT C C. perfringens CPerf165F for CGCATAACG TTGAAAGAT GGCPerf269R rev CCTTGGTAG GCCGTTACC C CPerf187T probe TCATCATTC AACCAAAGGAGCAATCC netB gene NetB-RJ- Fwd TGGTGCTGG Fwd AATAAATGC TTCAT NetB-RJ-Rev TGCATCATC Rev TTTTCTTTG AATTGTTC NetB-RJ- Probe ATACTATAA MGBGCTATGAAC AACC

Binding Assay to Characterize the Capability of Lactobacillus Isolatesto Attach to Mucin and Collagen

The major component of the mucus membrane in the small intestines ismucin and the ability to bind to mucin was used to evaluate theirability to adhere to GI track. The binding assay was carried out with 96wells in a sterile non-TC treated polystyrene plate. The initial stepwas the coating of the plate with mucin (type II or type III porcinemucin, Sigma). The mucin solution (1%) was prepared by stirring for afew hours till a homogenous suspension was obtained. The solution wasautoclaved before use. 120 μl of mucin solution was added to each 96well and the plate was incubated at 4° C. overnight. Right before use,the unbound mucin was removed and rinsed twice with 125 μl molecularbiology grade water.

For binding assay, fresh overnight cultures grown in MRS medium at 37 or40° C. were used. The cultures were first collected by centrifugationand washed once with 1×PBS medium. 100 μl culture at 1 OD (600 nm) wasadded into each well in the washed mucin coated plate and the plate wasincubated in the 37° C. anaerobic chamber or anaerobic jar for 2 hours.After incubation, the plate was washed with 100 μl 1X PBS twice and theattached cells in each well was stained by adding 100 μl of 0.1%solution of crystal violet (1% stock solution from Sigma) diluted inwater. The plate was allowed to sit for 5 to 10 min at room temperature.The unbound dye was removed and each well was washed three times with100 μl 1X PBS. To measure the dye bound to the attached cells, 100 μl of1X PBS was used to re-suspend the bound cells by pipetting up and down.Alternatively, 100 μl of ethanol was used to dissolve the dye. After 20min, the dye intensity was measured at 570 or 590 nm. The readingreflected the number of attached cells.

A similar procedure was used to evaluate the ability of Lactobacillusstrains to bind the Biocoat collagen IV plates from Corning. It has beenreported that collagen binding is one of the important virulence factorsfor C. perfringens (Vet Microbiol. 2015 Nov 18;180:299-303). One of thepotential beneficial properties of DFM can be the competitive binding ofthis extracellular matrix.

Antimicrobial Assay to Characterize Lactobacillus Isolates forAntimicrobial Activity Against C. Perfringens

Microplate based antimicrobial assay was performed using supernatants ofLactobacillus isolates from chicken GIT samples. Isolates wereinoculated in MRS liquid medium and grew at 37° C. for 2 to 3 daysanaerobically. Cultures were centrifuged at 4500 rpm for 5 min andsupernatants were collected in Costar 96 well plate and filtered. 20 or25 μL supernatant was used for assay.

The target pathogen C. perfringens from a fresh plate was inoculated inBHI medium at 37° C. and grew overnight anaerobically. C. perfringensculture was diluted into an OD (600 nm) of 0.1 with BHI medium. 175 or180 μL diluted C. perfringens culture was added to each well containing20 or 25 μL supernatant to a final volume of 200 μl. For the control, 20or 25 μL of MRS medium was used. The assay plate was incubated underanaerobic conditions at 37° C. for 18 hours and then OD (600 nm) wasmeasured. The amount of the growth relative to the control was used tomeasure the antimicrobial activity. Alternatively, the growth wasmonitored continuously using a microtiter reader.

Animal Model for Necrotic Enteritis Caused by C perfringens

A challenged diseased model for broiler chicken has been usedextensively (Front Microbiol. 2016, 7:1416; J Anim Sci Biotechnol. 2018,9:25; Poult Sci. 2018, Nov 18). In this experiment, chickens at day 9were first challenged with live 1×Eimeria vaccine (ADVENT® coccidiosisvaccine, Huvepharma, Inc., Lincoln, Nebr. 68528). Seventeen (17) birdswere in each of the experimental and control groups. The vaccine wasdiluted in water and each chicken received 1 ml orally. At day 11, eachchicken received 1 ml of pathogen cocktail orally. The pathogen cocktailconsisted of five Clostridium perfringens strains isolated from diseasedtissues. All strains contained the netB and tpel genes based on thegenome analysis. These strains were grown individually in cooked meatmedium (Sigma) overnight and the fresh cultures were mixed in equalvolume in a glove box to make up the cocktail. The cocktail was used thesame day to induce necrotic enteritis.

With this disease model, Lactobacillus isolates were evaluated for theirefficacy against C. perfringens infection. Lactobacillus strains weregrown in MRS medium under anaerobic conditions. Fresh cultures weremixed in equal volume and the mixed cell suspension for each consortiumwas aliquoted into serum bottles. The serum bottles were stored at 80°C. and thawed before use. The Lactobacillus consortia were fed to eachchicken daily by gavage starting at day 1. At day 15, chicken intestineswere collected for histopathology analysis and the mucosa samples wereused for DNA isolation as described supra.

Example 2: Binding and Antimicrobial Properties of Lactobacillus StrainsIsolated from Chicken Small Intestines

Table 6 summarized the in vitro assay results of Lactobacillus strainsfor their antimicrobial ability and their ability to bind to eithermucin or collagen as described per the methods of Example 1. While theactivities were strain-dependent, the supernatants from L. salivariusisolates had high antimicrobial activity. Those from L. reuteri hadlower antimicrobial activity, but quite a few had excellent ability tobind to collagen.

TABLE 6 Results of in vitro assays of Lactobacillus strains MucinCollagen Antimicrobial Activity² Description Isolates Binding¹ Binding¹(%) L. agilis H3 0.16 0.22 72 L. agilis A3 0.46 0.33 32 L. agilis D10.31 0.59 11 L. crispatus D3 0.48 0.51 45 L. crispatus E3 0.98 0.58 7 L.gallinarum E1 0.24 0.34 0 L. gallinarum H1 0.59 0.87 0 L. johnsonii C30.43 0.54 92 L. reuteri E2 0.20 0.43 46 L. reuteri S2 0.42 0.50 8 L.reuteri A2 0.85 0.72 1 L. reuteri S1 0.34 0.72 33 L. reuteri S3 0.360.58 6 L. reuteri C1 0.59 1.04 7 L. salivarius D2 0.59 0.47 94 L.salivarius H2 0.23 0.40 94 L. salivarius A1 0.33 0.34 90 L. salivariusC2 0.88 1.24 77 ¹The binding activity was measured as OD at 570 nm. ²Theantimicrobial activity was measured as percent inhibition relative tothe control.

Example 3: Animal Model for Necrotic Enteritis Caused by C. Perfringens

DFM candidates were evaluated for their efficacy against C. perfringensinfection in an animal model. For animal study, the lactobacillusstrains were grouped as consortia based on their phylogenetic diversityand representation of a broad spectrum of in vitro assay results. Theconsortia and their strain composition are listed in Table 7. Thesestrains were grown in MRS medium under anaerobic conditions and thefresh culture were mixed in equal volume inside the glovebox to make upa specific consortium for the animal trial. The mixed cell suspensionfor each consortium was aliquoted into serum bottles under anaerobicconditions inside a glove box. The serum bottles were stored at 80° C.and thawed before use. Each consortium was fed to chicken daily bygavage starting at day 1. At day 15, chicken intestines were collectedfor histopathology analysis and the mucosa samples were used for DNAisolation as described in Example 1.

TABLE 7 List of Lactobacillus strains used for animal study ConsortiumStrain 1 Strain 2 Strain 3 A L. salivarius A1 L. reuteri A2 L. agilis A3C L. reuteri C1 L. salivarius C2 L. johnsonii C3 D L. agilis D1 L.salivarius D2 L. crispatus D3 E L. gallinarum E1 L. reuteri E2 L.crispatus E3 H L. gallinarum H1 L. salivarius H2 L. agilis H3 S L.reuteri S1 L. reuteri S2 L. reuteri S3

The abundance of C. perfringens based on 16S analysis using samples fromday 15 as described in Example 1 is summarized in Table 8. In thecontrol group (disease model) where no Lactobacillus strains were used,necrotic enteritis in the small intestines was observed. Consistent withthis result, there was abundance of C. perfringens as quantified basedon 16S analysis and netB qPCR. Chicken feed with consortia E and C,showed similar high abundance of C. perfringens as the chicken in thecontrol group. Chicken from these two groups suffered from necroticenteritis. This suggests that these two consortia did not show efficacyagainst the expansion of the pathogen in the animal trial. On the otherhand, the abundance of C. perfringens was much lower in consortia A, D,H, and S. Histopathology indicated no sign of necrotic enteritis. Thisresult suggests these four consortia had efficacy in the prevention ofC. perfringens infection. From this animal study, it is clear that theefficacy against C. perfringens infection depended on the specificcomposition of the consortium since both consortia C and E had some ofthe same Lactobacillus species as consortia in A, D, H, and S

TABLE 8 Results of animal trials Consortium CP Abundance¹ netB qPCR²Necrotic Enteritis Control 0.257 0.204 Positive C 0.349 0.335 Positive E0.335 0.216 Positive A 0.013 0.026 Negative D 0.021 0.007 Negative H0.001 0.001 Negative S 0.001 0.001 Negative ¹CP Abundance = 16S CPsequence reads/Total 16S Sequence reads (Example 1) ²netB qPCR = qPCRnetB Copy number/qPCR Total 16S

Example 4: Large Scale Animal Trial to Assess Improved MortalityBenefits of Consortium

In February 2020, four commercial broiler chicken houses located in theUnited States were each stocked with approximately 56,000 birds(approximately 224,000 total birds were used in this study). Necroticenteritis affecting large-scale chicken production in the U.S. istypically most prevalent during the months of November through April.

Each house was divided into two halves; 50% of the birds weresupplemented with the Direct Fed Microbial (DFM; a combination of 3 L.reuteri strains S1, S2, and S3), the remaining 50% of each house wereused as unsupplemented controls. Each half of the house received watervia a different waterline. All birds were fed a typical U.S. corn/soybased pelleted feed ad libitum.

Birds were placed on Day 1 and slaughtered on Day 52. The DFM wassupplemented into the waterline via the medicator at a dose of 1.2×10⁸CFU/bird/day on days 17, 18, 19, 20 and 21 of production for three outof 4 houses and day 17 only for one house (day 17 onwards coincides withthe highest rate of necrotic enteritis outbreaks typically observed inthe field during this time of the year).

Flock mortality data for each of the houses was collected and is shownin FIG. 1. Control birds exhibited a combined mortality rate ofapproximately 11% whereas birds receiving DFM supplementation exhibitedmortality rates of 6.9% (i.e. a 61.5% total reduction in mortality overthe course of the study).

Survival probability analysis was conducted, and production cost savingswere calculated on a per million bird bases. To calculate the costsavings delivered by the DFM supplementation, the following parameterswere taken into consideration: weekly mortality, weekly feedconsumption, feed cost ($0.11 per lb), chick cost ($0.34 USD per chick),and approximate condemnations. In this study, DFM supplementation andthe associated reduction in mortality resulted in avoided losses ofapproximately $5769 USD or savings of $51,514 USD per million birds.

Example 5: Identification and Removal of Antimicrobial Resistance (AMR)Genes

This Example demonstrates the identification and removal ofantimicrobial resistance (AMR) genes from consortia members. AMR genes,particularly those associated with mobile genetic elements, are ofconcern because they can in some instances be transferred amongdifferent bacterial cells, thereby promoting antibiotic resistance. Toprevent the spread of these AMR markers and to comply with regulatoryrequirements. it may be beneficial to remove them from strains used asDFMs in livestock.

The strains from the different consortia were sequenced using acombination of the short-read sequencing technology Illumina andlong-read sequencing technology platforms such as Oxford NanoporeTechnology or PacBio. The reads from a strain were processed usingstandard trimming procedures and assembled into a sequence usingstandard tools such as SPAdes (Bankevich et al,. 2012, J Comput Biol19:455-477), Unicycler (Wick et al., 2017, PLoS Comput Biol13:e1005595), and Pilon (Walker et al., 2014, PLoS One 9:e112963). Thesequence was subsequently annotated using prokka (Seemann, 2014,Bioinformatics 30:2068-2069) or PATRIC (Wattam et al. 2018, Methods MolBiol 1704:79-101). Antimicrobial resistance (AMR) genes were identifiedusing CARD (Alcock et al., 2020, Nucleic Acids Res 48:D517-D525), theComprehensive Antibiotic Resistance Database, and PARTIC (Davis et al.,2016, Sci Rep 6:27930). Any AMR genes identified by these databases weresubsequently inspected for mobile genetic elements within thesurrounding 10 kb.

Strain S3 was Found to be Free of AMR Genes, While S1 and S2 Both HadTwo Different AMR Genes Associated with Mobile Genetic Elements

InuC, a transposon-mediated nucleotidyltransferase, and vatE, atransposon-mediated acetyltransferase. Both genes are next to atransposase. The AMR gene and transposase were found to be flanked by aset of indirect and direct repeats (Achard et al., 2005, AntimicrobAgents Chernother 49:2716-2719). Four copies of lnuC spread throughoutthe genome and one copy of vatE were identified in S1, while one copy ofeither gene was found in S2. This strain S2 also contained a tetW genethat was not associated with any mobile genetic elements within thesurrounding 10 kb of the genes.

Positions of the genes in S1 were identified based on PacBio sequence oflnuC (470,273 forward, 995,196 reverse, 1,149,997 forward, 1,775,176forward) and vatE (1,439,964) and in S2 based on the ONT sequence ofInuC (161,023 forward), vatE (457,661 forward) and tetW (818,067reverse).

These genes and their associated transposases are and were removed usingstandard molecular tools such as recombineering (Zhang et al., 2018. JBacteriol 200; Ozcam et al., 2019, Appl Environ Microbiol 85), or CRISPRbased methods (Oh & Van Pijkeren, 2014, Nucleic Acids Res 42: e131).

SEQUENCES Lactobacillus agilis strain H3 16S rRNA (SEQ ID NO: 1)AATGAGAGTTTGATCCTGGCTCAGGACGAACGCTG GCGGCGTGCCTAATACATGCAAGTCGAACGCTTTTTTCAATCATCGTAGCTTGCTACACCGATTGAAAAT TGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAAAAGAGGGGGATAACACTTGGAAACAGGT GCTAATACCGCATAACCATGATGACCGCATGGTCATTATGTAAAAGATGGTTTCGGCTATCACTTTTGGA TGGACCCGCGGCGTATTAACTTGTTGGTGGGGTAACGGCCTACCAAGGTAATGATACGTAGCCGAACTGA GAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTT CCACAATGGGCGCAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTG TTGTTAGAGAAGAACATGCGAGAGAGTAACTGTTCTTGTATTGACGGTATCTAACCAGAAAGCCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGA ACGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAATTGCATTGGAAACTGGAG GACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACA CCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGTTCGAAAGTGTGGGTAGCAAACAGGAT TAGATACCCTGGTAGTCCACACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCT GCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACAT CTTTTGACCATCTTAGAGATAAGATTTTCCCTTCGGGGACAAAATGACAGGTGGTGCATGGCTGTCGTCA GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAA GTTGGGCACTCTGGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCC CCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAACTCGCGAGGGCAAGCT AATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGT AATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGA GTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGTCTAAGGTGGGACAGATGATTGG GGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT Lactobacillus salivarius strain A1 16S rRNA(SEQ ID NO: 2) AATGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAAACT TTCTTACACCGAATGCTTGCATTCACCGTAAGAAGTTGAGTGGCGGACGGGTGAGTAACACGTGGGTAAC CTGCCTAAAAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATATCTCTAAGGATCGCATGATC CTTAGATGAAAGATGGTTCTGCTATCGCTTTTAGATGGACCCGCGGCGTATTAACTAGTTGGTGGGGTAA CGGCCTACCAAGGTGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGC CCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGC GTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACACGAGTGAGAGTAACTGTTC ATTCGATGACGGTATCTAACCAGCAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG GCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAACGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCT TCGGCTTAACCGGAGTAGTGCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATG TGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGA CGCTGAGGTTCGAAAGTGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAA TGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTA CGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTC GAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGATTAGGCTTTCCCTTCG GGGACAAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAAGTTGGGCACTCTGGCGAGACTGCCGGTGACAAACC GGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGA CGGTACAACGAGTCGCAAGACCGCGAGGTTTAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGG CTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCGCAA GGAGCCAGCCGTCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTG CGGCTGGATCACCTCCTTTLactobacillus agilis strain A3 16S rRNA (SEQ ID NO: 3)AATGAGAGTTTGATCCTGGCTCAGGACGAACGCTG GCGGCGTGCCTAATACATGCAAGTCGAACGCTTTTTTCAATCATCGTAGCTTGCTACACCGATTGAAAAT TGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAAAAGAGGGGGATAACACTTGGAAACAGGT GCTAATACCGCATAACCATGATGACCGCATGGTCATTATGTAAAAGATGGTTTCGGGTATCACTTTTGGA TGGACCCGCGGCGTATTAACTTGTTGGTGGGGTAACGGCCTACCAAGGTAATGATACGTAGCCGAACTGA GAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTT CCACAATGGGCGCAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTG TTGTTAGAGAAGAACATGCAGGAGAGTAACTGTTCTTGTATTGACGGTATCTAACCAGAAAGCCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGA ACGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAATTGCATTGGAAACTGGAG GACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACA CCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGTTCGAAAGTGTGGGTAGCAAACAGGAT TAGATACCCTGGTAGTCCACACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCT GCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACAT CTTTTGACCATCTTAGAGATAAGATTTTCCCTTCGGGGACAAAATGACAGGTGGTGCATGGCTGTCGTCA GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAA GTTGGGCACTCTGGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCC CCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAACTCGCGAGGGCAAGCT AATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGT AATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGA GTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGTCTAAGGTGGGACAGATGATTGG GGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT Lactobacillus salivarius strain D2 16S rRNA(SEQ ID NO: 4) AATGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAAACT TTCTTACACCGAATGCTTGCATTCACCGTAAGAAGTTGAGTGGCGGACGGGTGAGTAACACGTGGGTAAC CTGCCTAAAAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATATCTCTAAGGATCGCATGATC CTTAGATGAAAGATGGTTCTGCTATCGCTTTTAGATGGACCCGCGGCGTATTAACTAGTTGGTGGGGTAA CGGCCTACCAAGGTGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGC CCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGC GTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACACGAGTGAGAGTAACTGTTC ATTCGATGACGGTATCTAACCAGCAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG GCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAACGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCT TCGGCTTAACCGGAGTAGTGCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATG TGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGA CGCTGAGGTTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAA TGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTA CGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGGATGTGGTTTAATTC GAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGATTAGGCTTTCCCTTCG GGGACAAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAAGTTGGGGACTCTGGCGAGACTGCCGGTGACAAACC GGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGA CGGTACAACGAGTCGCAAGACCGCGAGGTTTAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGG CTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCGCAA GGAGCCAGCCGTCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTG CGGCTGGATCACCTCCTTTLactobacillus agilis strain D1 16S rRNA (SEQ ID NO: 5)AATGAGAGTTTGATCCTGGCTCAGGACGAACGCTG GCGGCGTGCCTAATACATGCAAGTCGAACGCTTTTTTCAATCATCGTAGCTTGCTACACCGATTGAAAAT TGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAAAAGAGGGGGATAACACTTGGAAACAGGT GCTAATACCGCATAACCATGATGACCGCATGGTCATTATGTAAAAGATGGTTTCGGCTATCACTTTTGGA TGGACCCGCGGCGTATTAACTTGTTGGTGGGGTAACGGCCTACCAAGGTAATGATACGTAGCCGAACTGA GAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTT CCACAATGGGCGCAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTG TTGTTAGAGAAGAACATGCAGGAGAGTAACTGTTCTTGTATTGACGGTATCTAACCAGAAAGCCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGA ACGCAGGCGGTCCTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAATTGCATTGGAAACTGGAG GACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACA CCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGTTCGAAAGTGTGGGTAGCAAACAGGAT TAGATACCCTGGTAGTCCACACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCT GCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACAT CTTTTGACCATCTTAGAGATAAGATTTTCCCTTCGGGGACAAAATGACAGGTGGTGCATGGCTGTCGTCA GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAA GTTGGGCACTCTGGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCC CCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAACTCGCGAGGGCAAGCT AATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGT AATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGA GTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGTCTAAGGTGGGACAGATGATTGG GGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT Lactobacillus crispatus strain D3 16S rRNA(SEQ ID NO: 6) AATGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCGG AACTAACAGATTTACTTCGGTAATGACGTTAGGAAAGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAA CCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGAT CAGCTTTTAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGT AAAGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACG GCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCC GCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGG CCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGG TGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGC CCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCA TGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACT GACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATG AGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAG TACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT TCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGCCATTTGTAGAGATACAAAGTTCCCTT CGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTTGTTATTAGTTGCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAA CCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATG GGCAGTACAACGAGAAGCGAGCCTGCGAAGGCAAGCGAATCTCTGAAAGCTGTTCTCAGTTCGGACTGCA GTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTT CCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTCTGCAATGCCCAAAGCCGGTGGCCTAACCTT CGGGAAGGAGCCGTCTAAGGCAGGGCAGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACC TGCGGCTGGATCACCTCCTTTLactobacillus reuteri strain S1 16S rRNA (SEQ ID NO: 7)GTGACGGTATCCAACCAGAAAGTCACGGCTAACTA CGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCA GGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTT GAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGT GGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGAT ACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGC TAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCC GCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGC GCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCG TGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGG GCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTA TGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCT CTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCG CGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTG TAACGCCCAAAGTCGGTGGCCTAACCTTTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTGA AGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT Lactobacillus reuteri strain S2 16S rRNA (SEQ ID NO: 8)GTGACGGTATCCAACCAGAAAGTCACGGCTAACTA CGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCA GGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTT GAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGT GGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGAT ACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGC TAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCC GCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGC GCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCG TGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGG GCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTA TGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCT CTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCG CGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTG TAACGCCCAAAGTCGGTGGCCTAACCTTTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTGA AGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT Lactobacillus reuteri strain S3 16S rRNA (SEQ ID NO: 9)TTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGA TGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGA GAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTT CCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTG TTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGA GCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGC GACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACA CCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGAT TAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCC GGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGA CATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCAT TAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCAT GCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAA GCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCT AGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATG GGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCTTTATGGAGGGAGCCGCCTAAGGCGGGACAGATGAC TGGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT Lactobacillus saliva tins strain H2 16S TRNA(SEQ ID NO: 10) AATGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAAACT TTCTTACACCGAATGCTTGCATTCACCGTAAGAAGTTGAGTGGCGGACGGGTGAGTAACACGTGGGTAAC CTGCCTAAAAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATATCTCTAAGGATCGCATGATC CTTAGATGAAAGATGGTTCTGCTATCGCTTTTAGATGGACCCGCGGCGTATTAACTAGTTGGTGGGGTAA CGGCCTACCAAGGTGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGC CCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGC GTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACACGAGTGAGAGTAACTGTTC ATTCGATGACGGTATCTAACCAGCAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG GCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAACGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCT TCGGCTTAACCGGAGTAGTGCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATG TGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGA CGCTGAGGTTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAA TGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTA CGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTC GAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGATTAGGCTTTCCCTTCG GGGACAAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAAGTTGGGCACTCTGGCGAGACTGCCGGTGACAAACC GGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGA CGGTACAACGAGTCGCAAGACCGCGAGGTTTAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGG CTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCGCAA GGAGCCAGCCGTCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTG CGGCTGGATCACCTCCTTTLactobacillus gallinarum strain Hl 16S rRNA (SEQ ID NO: 11)AATGAGAGTTTGATCCTGGCTCAGGACGAACGCTG GCGGCGTGCCTAATACATGCAAGTCGAGCGAGCAGAACCAGCAGATTTACTTCGGTAATGACGCTGGGGA CGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAG GTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTATAAAAGGCGGCGTAAGCTGTCGCTATGG GATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTT GAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATC TTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTC TGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGC GAGCGCAGGCGGAAAAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGT TTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAA CACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGG ATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTG CTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACG GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGAC ATCTAGTGCCATCCTAAGAGATTAGGAGTTCCCTTCGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATTAGTTGCCAGCATT AAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATG CCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGTACAACGAGAAGCGAGCCTGCGAAGGCAAG CGAATCTCTGAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTA GTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGG AAGTCTGCAATGCCCAAAGCCGGTGGCCTAACCTTCGGGAAGGAGCCGTCTAAGGCAGGGCAGATGACTG GGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT Lactobacillus reuteri strain A2 16S rRNA(SEQ ID NO: 12) TATGAGAGTTTGATCCTGGCTCAGGATGAACGCCGGCGGTGTGCCTAATACATGCAAGTCGTACGCACTG GCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAA CACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAA AGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTA GTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGA ACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGAT GGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTG AGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGG TAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCT GATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACA GTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCT GGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGC CGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTC CGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAA GGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGG TTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTG CCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACAC GTGCTACAATGGACGGTACAACGAGTCGCAAGCTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAG TTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCG GTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGT GGCCTAACCTTTATGGAGGGAGCCGCCTAAGGCGGGACAGATGACTGGGGTGAAGTCGTAACAAGGTAGC CGTAGGAGAACCTGCGGCTGGATCACCTCCTTT

We claim:
 1. A feed additive composition comprising a direct fedmicrobial (DFM) comprising (a) at least one biologically pure strain of(i) Lactobacillus reuteri; and/or (ii) L. salivarius; and (b) one ormore additional biologically pure strain(s) of (i) L. reuteri; (ii) L.agilis; (iii) L. crispatus; and/or (iv) L. gallinarum.
 2. The feedadditive composition of claim 1, comprising three biologically purestrains of L. reuteri.
 3. The feed additive composition of claim 1 orclaim 2, comprising (a) a bacterial strain having a 16S ribosomal RNAsequence displaying at least 97.0% sequence similarity to a 16Sribosomal RNA sequence of a L. reuteri strain S1 deposited at WesterdijkFungal Biodiversity Institute (WFDB) under number CBS 145921; and (b)(i)a bacterial strain having a 16S ribosomal RNA sequence displaying atleast 97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.reuteri strain S2 deposited at WFDB under number CBS 145922; and (ii) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.reuteri strain S3 deposited at WFDB under number CBS
 145923. 4. The feedadditive composition of any one of claims 1-3, wherein the compositioncomprises (a) L. reuteri strain S1 (CBS 145921) or a live strain havingall of the identifying characteristics of L. reuteri strain S1 (CBS145921); and (b)(i) L. reuteri strain S2 (CBS 145922) or a live strainhaving all of the identifying characteristics of L. reuteri strain S2(CBS 145922); and (ii) L. reuteri strain S3 (CBS 145923) or a livestrain having all of the identifying characteristics of L. reuteristrain S3 (CBS 145923) either (A) alone; or (B) in combination with aculture supernatant derived from each of these strains.
 5. The feedadditive composition of claim 1, comprising (a) a biologically purestrain of L. salivarius; and (b)(i) a biologically pure strain of L.gallinarum; and (ii)(A) a biologically pure strain of L. agilis; or (B)a biologically pure strain of L. reuteri.
 6. The feed additivecomposition of claim 1 or claim 5, comprising (a) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of a L. salivarius strain H2deposited at WFDB under number CBS 145919; and (b)(i) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of a L. gallinarum strain H1deposited at WFDB under number CBS 145918; and (ii)(A) a bacterialstrain having a 16S ribosomal RNA sequence displaying at least 97.0%sequence similarity to a 16S ribosomal RNA sequence of an L. agilisstrain H3 comprising SEQ ID NO:1; or (B) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of an L. reuteri strain A2 deposited atWFDB under number CBS
 145924. 7. The feed additive composition of claim6, wherein the composition comprises (a) L. salivarius strain H2 (CBS145919) or a live strain having all of the identifying characteristicsof L. salivarius strain H2 (CBS 145919); and (b)(i) L. gallinarum strainH1 (CBS 145918) or a live strain having all of the identifyingcharacteristics of L. gallinarum strain H1 (CBS 145918); and (ii) L.reuteri strain A2 (CBS 145924) or a live strain having all of theidentifying characteristics of L. reuteri strain A2 (CBS 145924) either(A) alone; or (B) in combination with a culture supernatant derived fromeach of these strains.
 8. The feed additive composition of claim 1,comprising (a) a biologically pure strain of L. salivarius; and (b)(i) abiologically pure strain of L. agilis; and a biologically pure strain ofL. reuteri.
 9. The feed additive composition of claim 1 or claim 8,comprising (a) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of an L. salivarius strain A1 comprising SEQ ID NO:2; and(b)(i) a bacterial strain having a 16S ribosomal RNA sequence displayingat least 97.0% sequence similarity to a 16S ribosomal RNA sequence of anL. agilis strain A3 comprising SEQ ID NO:3; and (ii) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of an L. reuteri strain A2deposited at WFDB under number CBS 145924 either (A) alone; or (B) incombination with a culture supernatant derived from each of thesestrains.
 10. The feed additive composition of claim 9, comprising L.reuteri strain A2 (CBS 145924) or a live strain having all of theidentifying characteristics of L. reuteri strain A2 (CBS 145924). 11.The feed additive composition of claim 1, comprising (a) a biologicallypure strain of L. salivarius; and (b)(i) a biologically pure strain ofL. agilis; and (ii) a biologically pure strain of L. crispatus.
 12. Thefeed additive composition of claim 1 or claim 11, comprising (a) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.salivarius strain D2 comprising SEQ ID NO:4; (b)(i) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of an L. agilis strain D1comprising SEQ ID NO:5; and (ii) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of an L. crispatus strain D3 comprising SEQID NO:6 either (A) alone; or (B) in combination with a culturesupernatant derived from each of these strains.
 13. The feed additivecomposition of claim 3 or claim 4, wherein a) the 16S ribosomal RNAsequence of L. reuteri strain S1 comprises the nucleotide sequence ofSEQ ID NO:7; and (b)(i) the 16S ribosomal RNA sequence of L. reuteristrain S2 comprises the nucleotide sequence of SEQ ID NO:8; and (ii) the16S ribosomal RNA sequence of L. reuteri strain S3 comprises thenucleotide sequence of SEQ ID NO:9.
 14. The feed additive composition ofclaim 6 or claim 7, wherein a) the 16S ribosomal RNA sequence of L.salivarius strain H2 comprises the nucleotide sequence of SEQ ID NO:10;and (b)(i) the 16S ribosomal RNA sequence of L. gallinarum strain H1comprises the nucleotide sequence of SEQ ID NO:11; and (ii) the 16Sribosomal RNA sequence of L. reuteri strain A2 comprises the nucleotidesequence of SEQ ID NO:12.
 15. The feed additive composition of claim 9,wherein (b)(ii) the 16S ribosomal RNA sequence of L. reuteri strain A2comprises the nucleotide sequence of SEQ ID NO:12.
 16. The feed additivecomposition of any one of claims 1-15, wherein said one or more strainsfurther comprise one or more inactivated or deleted antimicrobialresistance (AMR) genes.
 17. The feed additive composition of any one ofclaims 1-16, wherein the composition produces one or more organic acidsselected from the group consisting of lactate, butyrate, isobutyrate,propionate, acetate, isovalerate, and valerate.
 18. The feed additivecomposition of any one of claims 1-17, further comprising one or moreenzymes.
 19. The feed additive composition of claim 18, wherein the oneor more enzymes are selected from the group consisting of a phytase, aprotease, an amylase, a xylanase, and a beta-glucanase.
 20. The feedadditive composition of any one of claims 1-19, further comprising oneor more essential oils.
 21. The feed additive composition of any one ofclaims 1-20, wherein each strain is present at a concentration of atleast about 1×10³ CFU/g feed additive composition to at least about1×10¹⁵ CFU/g feed additive composition.
 22. The feed additivecomposition of any one of claims 1-21, wherein the composition inhibitsat least one pathogen selected from avian pathogenic Salmonella sp.,Escherichia coli, Clostridium perfringens and Enterobacteriaceae in agastrointestinal tract of a bird having ingested an effective amount ofsaid direct fed microbial composition.
 23. The feed additive compositionof any one of claims 1-22, wherein the composition is formulated fordelivery to an animal via waterline.
 24. A bacterial consortiumcomprising (a) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of a L. reuteri strain S1 deposited at Westerdijk FungalBiodiversity Institute (WFDB) under number CBS 145921; and (b)(i) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.reuteri strain S2 deposited at WFDB under number CBS 145922; and (ii) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.reuteri strain S3 deposited at WFDB under number CBS
 145923. 25. Theconsortium of claim 24, comprising (a) L. reuteri strain S1 (CBS 145921)or a live strain having all of the identifying characteristics of L.reuteri strain S1 (CBS 145921); and (b)(i) L. reuteri strain S2 (CBS145922) or a live strain having all of the identifying characteristicsof L. reuteri strain S2 (CBS 145922); and (ii) L. reuteri strain S3 (CBS145923) or a live strain having all of the identifying characteristicsof L. reuteri strain S3 (CBS 145923) either (A) alone; or (B) incombination with a culture supernatant derived from each of thesestrains.
 26. The consortium of claim 24 or claim 25, wherein a) the 16Sribosomal RNA sequence of L. reuteri strain S1 comprises the nucleotidesequence of SEQ ID NO:7; and (b)(i) the 16S ribosomal RNA sequence of L.reuteri strain S2 comprises the nucleotide sequence of SEQ ID NO:8; and(ii) the 16S ribosomal RNA sequence of L. reuteri strain S3 comprisesthe nucleotide sequence of SEQ ID NO:9.
 27. A bacterial consortiumcomprising (a) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of a L. salivarius strain H2 deposited at WFDB under number CBS145919; and (b)(i) a bacterial strain having a 16S ribosomal RNAsequence displaying at least 97.0% sequence similarity to a 16Sribosomal RNA sequence of a L. gallinarum strain H1 deposited at WFDBunder number CBS 145918; and (ii)(A) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of an L. agilis strain H3 comprising SEQ IDNO:1; or (B) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of an L. reuteri strain A2 deposited at WFDB under number CBS145924.
 28. The consortium of claim 27, comprising (a) L. salivariusstrain H2 (CBS 145919) or a live strain having all of the identifyingcharacteristics of L. salivarius strain H2 (CBS 145919); and (b)(i) L.gallinarum strain H1 (CBS 145918) or a live strain having all of theidentifying characteristics of L. gallinarum strain H1 (CBS 145918); and(ii) L. reuteri strain A2 (CBS 145924) or a live strain having all ofthe identifying characteristics of L. reuteri strain A2 (CBS 145924)either (A) alone; or (B) in combination with a culture supernatantderived from each of these strains.
 29. A bacterial consortiumcomprising (a) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of an L. salivarius strain A1 comprising SEQ ID NO:2; and(b)(i) a bacterial strain having a 16S ribosomal RNA sequence displayingat least 97.0% sequence similarity to a 16S ribosomal RNA sequence of anL. agilis strain A3 comprising SEQ ID NO:3; and (ii) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of an L. reuteri strain A2deposited at WFDB under number CBS 145924 either (A) alone; or (B) incombination with a culture supernatant derived from each of thesestrains.
 30. The consortium of claim 29, comprising L. reuteri strain A2(CBS 145924) or a live strain having all of the identifyingcharacteristics of L. reuteri strain A2 (CBS 145924).
 31. A bacterialconsortium comprising (a) a bacterial strain having a 16S ribosomal RNAsequence displaying at least 97.0% sequence similarity to a 16Sribosomal RNA sequence of an L. salivarius strain D2 comprising SEQ IDNO:4; (b)(i) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of an L. agilis strain D1 comprising SEQ ID NO:5; and (ii) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.crispatm strain D3 comprising SEQ ID NO:6 either (A) alone; or (B) incombination with a culture supernatant derived from each of thesestrains.
 32. The bacterial consortium of any one of claims 24-31,wherein one or more bacterial strains further comprise one or moreinactivated or deleted Antimicrobial resistance (AMR) genes.
 33. Thebacterial consortium of any one of claims 24-32, wherein the consortiumproduces one or more organic acids selected from the group consisting oflactate, butyrate, isobutyrate, propionate, acetate, isovalerate, andvalerate.
 34. The bacterial consortium of any one of claims 24-33,wherein each strain is present at a concentration of at least about1×10³ CFU/animal/day to at least about 1×10¹⁵ CFU/animal/day in theconsortium.
 35. The bacterial consortium of any one of claims 24-34,wherein the consortium inhibits at least one pathogen selected fromavian pathogenic Salmonella sp., Escherichia coli, Clostridiumperfringens and Enterobacteriaceae in a gastrointestinal tract of a birdhaving ingested an effective amount of said direct fed microbialcomposition.
 36. A premix comprising the feed additive composition ofany one of claims 1-23 or the bacterial consortium of any one of claims24-34 and at least one mineral and/or at least one vitamin.
 37. A feedcomprising the feed additive compositions of any one of claims 1-23 orthe bacterial consortium of any one of claims 24-34 or the premix ofclaim
 36. 38. A kit comprising a)(i) the feed additive composition ofany one of claims 1-23; (ii) the bacterial consortium of any one ofclaims 24-34; or (iii) the premix of claim 36; and b) writteninstructions for administration to an animal.
 39. The kit of claim 38,further comprising one or more enzymes.
 40. The kit of claim 39, whereinthe one or more enzymes are selected from the group consisting of aphytase, a protease, an amylase, a xylanase and a beta-glucanase.
 41. Amethod for improving one or more metrics in an animal selected from thegroup consisting of increased bodyweight gain, intestinal health status,decreased feed conversion ratio (FCR), improved gut barrier integrity,reduced mortality, reduced pathogen infection, and reduced pathogenshedding in feces comprising administering an effective amount of thefeed additive composition of any one of claims 1-23, the bacterialconsortium of any one of claims 24-34, the premix of claim 36, or thefeed of claim 37 to the animal, thereby improving the one or moremetrics in the animal.
 42. The method of claim 41, wherein the feedadditive composition increases one or more of the lactate, acetate,isobutyrate, butyrate, isovalerate, and/or valerate content of thegastrointestinal tract of the animal.
 43. The method of claim 41 orclaim 42, wherein the pathogen is one or more of Clostridiumperfringens, Campylobacter jejuni, Enterobacteriaceae, a Salmonella sp.,and/or Escherichia coli.
 44. The method of any one of claims 41-43,wherein the method further treats, prevents, or decreases incidence ofnecrotic enteritis.
 45. The method of any one of claims 41-44, whereinthe animal is a domesticated bird.
 46. The method of claim 45, whereinthe domesticated bird is selected from the group consisting of chickens,turkeys, ducks, geese, quail, emus, ostriches, and pheasant.
 47. Themethod of claim 46, wherein the chicken is a broiler or a layer.
 48. Themethod of any one of claims 41-47, wherein the feed additivecomposition, the bacterial consortium, or the premix is administered bywaterline.
 49. A method for preparing a feed additive composition or abacterial consortia comprising combining (a) a bacterial strain having a16S ribosomal RNA sequence displaying at least 97.0% sequence similarityto a 16S ribosomal RNA sequence of a L. reuteri strain S1 deposited atWesterdijk Fungal Biodiversity Institute (WFDB) under number CBS 145921;and (b)(i) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of a L. reuteri strain S2 deposited at WFDB under number CBS145922; and (ii) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of a L. reuteri strain S3 deposited at WFDB under number CBS145923.
 50. The method of claim 49, wherein (a) the L. reuteri strain S1is a L. reuteri strain S1 (CBS 145921) or a live strain having all ofthe identifying characteristics of L. reuteri strain S1 (CBS 145921);and (b)(i) the L. reuteri strain S2 is a L. reuteri strain S2 (CBS145922) or a live strain having all of the identifying characteristicsof L. reuteri strain S2 (CBS 145922); and (ii) the L. reuteri strain S3is a L. reuteri strain S3 (CBS 145923) or a live strain having all ofthe identifying characteristics of L. reuteri strain S3 (CBS 145923)either (A) alone; or (B) in combination with a culture supernatantderived from each of these strains.
 51. A method for preparing a feedadditive composition or a bacterial consortia comprising combining (a) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of a L.salivarius strain H2 deposited at WFDB under number CBS 145919; and(b)(i) a bacterial strain having a 16S ribosomal RNA sequence displayingat least 97.0% sequence similarity to a 16S ribosomal RNA sequence of aL. gallinarum strain H1 deposited at WFDB under number CBS 145918; and(ii)(A) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of an L. agilis strain H3 comprising SEQ ID NO:1; or (B) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.reuteri strain A2 deposited at WFDB under number CBS
 145924. 52. Themethod of claim 51, wherein (a) the L. salivarius strain H2 is an L.salivarius strain H2 (CBS 145919) or a live strain having all of theidentifying characteristics of L. salivarius strain 112 (CBS 145919);and (b)(i) the L. gallinarum strain H1 is an L. gallinarum strain H1(CBS 145918) or a live strain having all of the identifyingcharacteristics of L. gallinarum strain H1 (CBS 145918): and (ii) the L.reuteri strain A2 is an L. reuteri strain A2 (CBS 145924) or a livestrain having all of the identifying characteristics of L. reuteristrain A2 (CBS 145924) either (A) alone; or (B) in combination with aculture supernatant derived from each of these strains.
 53. A method forpreparing a feed additive composition or a bacterial consortiacomprising combining (a) a bacterial strain having a 16S ribosomal RNAsequence displaying at least 97.0% sequence similarity to a 16Sribosomal RNA sequence of an L. salivarius strain A1 comprising SEQ IDNO:2; and (b)(i) a bacterial strain having a 16S ribosomal RNA sequencedisplaying at least 97.0% sequence similarity to a 16S ribosomal RNAsequence of an L. agilis strain A3 comprising SEQ ID NO:3; and (ii) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.reuteri strain A2 deposited at WFDB under number CBS 145924 either (A)alone; or (B) in combination with a culture supernatant derived fromeach of these strains.
 54. The method of claim 53, wherein, the L.reuteri strain A2 is a L. reuteri strain A2 (CBS 145924) or a livestrain having all of the identifying characteristics of L. reuteristrain A2 (CBS 145924).
 55. A method for preparing a feed additivecomposition or a bacterial consortia comprising combining (a) abacterial strain having a 16S ribosomal RNA sequence displaying at least97.0% sequence similarity to a 16S ribosomal RNA sequence of an L.salivarius strain D2 comprising SEQ ID NO:4; (b)(i) a bacterial strainhaving a 16S ribosomal RNA sequence displaying at least 97.0% sequencesimilarity to a 16S ribosomal RNA sequence of an L. agilis strain D1comprising SEQ ID NO:5; and (ii) a bacterial strain having a 16Sribosomal RNA sequence displaying at least 97.0% sequence similarity toa 16S ribosomal RNA sequence of an L. crispatus strain D3 comprising SEQID NO:6 either (A) alone; or (B) in combination with a culturesupernatant derived from each of these strains.
 56. The method of anyone of claims 49-55, further comprising combining one or more enzyme(s)with the feed additive composition.
 57. The method of claim 56, whereinthe one or more enzymes are selected from the group consisting of aphytase, a protease, an amylase, a xylanase and a beta-glucanase. 58.The method of any one of claims 49-55, wherein (a) at least about 1×10³CFU/g feed additive composition to at least about 1×10¹⁵ CFU/g feedadditive composition is combined to form the feed additive composition:or (b) at least about 1×10³ CFU/animal/day bacterial consortium to atleast about 1×10¹⁵ CFU/animal/day is combined to form the bacterialconsortium.
 59. The method of any one of claims 49-58, furthercomprising packaging the feed additive composition.
 60. A method forpreparing a premix comprising combining the feed additive composition ofany one of claims 1-23 with at least one mineral and/or at least onevitamin.
 61. The method of claim 60, further comprising packaging thepremix.
 62. A method for removing one or more antimicrobial resistance(AMR) genes from a microbial strain comprising: (a) sequencing thegenome of one or more of the bacterial strains of the consortium of anyone of claims 24-31 to identify the presence of one or more AMR genes;(b) removing or deactivating said one or more AMR genes from the genomeof the one or more bacterial strains.
 63. The method of claim 62,wherein the bacterial strains are selected from the group consisting ofL. reuteri strain S1 (CBS 145921) or a live strain having all of theidentifying characteristics of L. reuteri strain S1 (CBS 145921) and L.reuteri strain S2 (CBS 145922) or a live strain having all of theidentifying characteristics of L. reuteri strain S2 (CBS 145922). 64.The method of claim 62 or claim 63, wherein the AMR gene is one or moregenes selected from the group consisting of InuC, vatE, and tetW. 65.The method of any one of claims 62-64, wherein the AMR gene is locatedwithin 10 kb of a transposon or other mobile genetic element.